Here we describe that InsP7, which is synthesized by InsP6K, competes with Akt PH-domain for the binding of PtdIns(3,4,5)P3 and thus negatively regulates PtdIns(3,4,5)P3-mediated cellular functions in neutrophils. Disruption of InsP6K1, one of the three mammalian InsP6 kinases that convert InsP6 to InsP7, resulted in enhanced PtdIns(3,4,5)P3 signaling in murine neutrophils. InsP6K1-deficient neutrophils exhibited elevated phagocytic and bactericidal capabilities as well as amplified NADPH oxidase-mediated superoxide production.
InsP7 contains a high energy pyrophosphate and has been implicated in a variety of cellular activities, but its signaling pathways have been obscure. Our observation that InsP7 is a physiologic regulator of interaction between Akt PH domain and PtdIns(3,4,5)P3 may be indicative of a general phenomenon. InsP7 functions as a PtdIns(3,4,5)P3-binding competitor for several other PH-domain containing proteins, such as PIKE and Tiam16
. InsP7 can also bind and regulate the function of proteins without a PH domain. InsP7 directly binds cytosolic cyclin-dependent kinase (CDK)-CDK inhibitor (CKI) complex, required for phosphate homeostasis in yeast41
. Additionally, it binds to clathrin-associated proteins such as AP2 and AP18042, 43
. Binding of InsP7 to AP180 negatively regulates clathrin cage assembly activity43, 44
. InsP7 can also serve as a phosphate donor, in a nonenzymatic fashion45, 46
. Inositol pyrophosphates can only transfer their high-energy β-phosphate moiety to pre-phosphorylated serine residues to generate pyrophosphoserine45, 46
. InsP7-mediated protein phosphorylation occurs predominantly at a region containing extensive stretches of serine residues surrounded by acidic amino acids45, 46
. Such sequence was not found in Akt, which does not seem to be a target protein45, 46
. Thus pyrophosphorylation is unlikely involved in the regulation of Akt by InsP7.
Activation of neutrophils at inflammatory sites is an essential component of the innate immune response. However, hyper-activation of neutrophils can also damage surrounding tissues via the release of toxic reactive oxygen species and granule enzymes such as proteases, causing acute inflammation. Thus, neutrophil activity needs to be carefully controlled by both positive and negative regulators. InsP7 becomes a putative candidate for such homeostatic regulation. The amount of InsP7 is tightly regulated in neutrophils. A substantial amount of InsP7 exists in unstimulated cells, preventing neutrophil hyperactivation and ensuring optimal cellular inflammatory response. InsP7 is rapidly reduced upon chemoattractant stimulation, allowing the induction of sustained PtdIns(3,4,5)P3 signals in responding neutrophils. InsP7 negatively regulates Akt signaling in glucose homeostasis and protein translation15
but, unlike the observations in the present study, where chemoattractant inhibits InsP7 formation, growth factors stimulate InsP7 generation. Therefore, although the inhibition of Akt signaling by InsP7 may be a general phenomenon in cellular signal transduction, the mechanism of its regulation as well as the resulting physiological consequences can be considerably different in different cell systems. Currently, the mechanisms by which InsP7 production is suppressed in chemoattractant stimulated neutrophils are largely unknown. It likely involves activation of inositol pyrophosphate phosphatase and/or deactivation of InsP6 kinase.
PtdIns(3,4,5)P3 signaling was implicated in several cellular processes related to neutrophil trafficking, particularly adhesion and chemotactic migration18, 38–40
. Similar results were also reported in other cell types such as mast cells, in which PI3K pathway plays an important role in integrin-mediated cell adhesion and migration47
. Nevertheless, InsP6K1 disruption failed to further augment cell adhesion, directionality, and migration speed in neutrophils. These results are somewhat different from the migration phenotypes observed in PTEN-deficient neutrophils, which also exhibit markedly enhanced PtdIns(3,4,5)P3 signaling21
. Although the overall chemotactic migration is relatively normal, PTEN disruption results in mildly impaired directionality, enhanced sensitivity to chemoattractant stimulation and slightly increased migration speed21
. The distinct effects are likely caused by different temporal and spatial regulation of PTEN and InsP6K1 in neutrophils. PTEN activity is increased and its subcellular localization is altered after chemoattractant stimulation48
. On the contrary, InsP7 level is high in unstimulated neutrophils and is significantly reduced after chemoattractant stimulation. In addition, the mechanisms by which PTEN and InsP6K1 regulate PtdIns(3,4,5)P3 signaling are different. PTEN regulates the amount of PtdIns(3,4,5)P3 and controls neutrophil function via
several downstream pathways. By contrast, InsP6K1 deletion does not alter the amount of PtdIns(3,4,5)P3 in the cell and its effect is limited to inhibition of Akt.
Although increased PtdIns(3,4,5)P3 signaling following InsP6K1 deletion directly improved the phagocytic and bactericidal capability of neutrophils, we cannot completely rule out that other cell types, such as macrophages, also account for the improved bacterial killing in the peritoneum of InsP6K1−/−
mice. The enhanced bacterial killing in the InsP6K1−/−
mice is associated with attenuated peritoneal neutrophil accumulation. It is unlikely that this effect is caused by accelerated neutrophil death, because we measured the neutrophil numbers at 4 hr after the induction of peritonitis, when neutrophil death has not yet occurred. We also directly examined neutrophil spontaneous death using an in vitro
assay and found no significant difference between wild-type and InsP6K1−/−
neutrophils. Additionally, using an adoptive transfer assay, we revealed that disruption of InsP6K1 does not affect neutrophil migration to sites of inflammation. Thus, the reduced neutrophil accumulation is likely an outcome of augmented bacteria clearance and accelerated resolution of inflammation. Alternatively, it can be due to the elevated ROS level in the inflamed peritoneal cavity of InsP6K1-deficient mice. ROS are able to deactivate proinflammatory chemokines such as C5a, fMLP35
, and IL837
, leading to reduced neutrophil recruitment.
Our findings indicate that higher inositol phosphates are significant players in a variety of cellular functions and can regulate signal transduction in a ubiquitous fashion akin to well-described signaling effectors such as kinases and phosphatases. The fairly ubiquitous expression of InsP6K1 suggests it may also regulate other blood cells such as lymphocytes and macrophages. These findings suggest that InsP6K1 and its phosphorylation product InsP7 may be promising therapeutic targets for modulating immune cell functions in various infectious and inflammatory diseases.