p53 ensures the normal behavior and modification of G1/S-specific histone H3.1 in the nucleus.

H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here, we found that p53 is necessary to secure the normal behavior and modification of H3.1 in the nucleus during the G1/S phase, in which p53 increases C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1) levels and decreases enhancer of zeste homolog 2 (EZH2) levels in the H3.1 interactome. In the absence of p53, H3.1 molecules tended to be tethered at or near the nuclear envelope (NE), where they were predominantly trimethylated at lysine 27 (H3K27me3) by EZH2, without forming nucleosomes. This accumulation was likely caused by the high affinity of H3.1 toward phosphatidic acid (PA). p53 reduced nuclear PA levels by increasing levels of CTDNEP1, which activates lipin to convert PA into diacylglycerol. We moreover found that the cytosolic H3 chaperone HSC70 attenuates the H3.1-PA interaction, and our molecular imaging analyses suggested that H3.1 may be anchored around the NE after their nuclear entry. Our results expand our knowledge of p53 function in regulation of the nuclear behavior of H3.1 during the G1/S phase, in which p53 may primarily target nuclear PA and EZH2.

A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

PLEKHS1 drives PI3Ks and remodels pathway homeostasis in PTEN-null prostate.

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression.

Attenuated cerebellar phenotypes in Inpp4a truncation mutants with preserved phosphatase activity.

Phosphoinositides (PIPs) act as intracellular signaling molecules that regulate various cellular processes. Abnormalities in PIP metabolism cause various pathological conditions, including neurodegenerative diseases, cancer and immune disorders. Several neurological diseases with diverse phenotypes, such as ataxia with cerebellar atrophy or intellectual disability without brain malformation, are caused by mutations in INPP4A, which encodes a phosphoinositide phosphatase. We examined two strains of Inpp4a mutant mice with distinct cerebellar phenotypes: the Inpp4aΔEx1,2 mutant exhibited striatal degeneration without cerebellar atrophy, and the Inpp4aΔEx23 mutant exhibited a severe striatal phenotype with cerebellar atrophy. Both strains exhibited reduced expression of Inpp4a mutant proteins in the cerebellum. N-terminal-truncated Inpp4a proteins were expressed from the Inpp4aΔEx1,2 allele by alternative translation initiation and had phosphatase activity for PI(3,4)P2, whereas the Inpp4a mutant protein encoded by Inpp4aΔEx23 completely lacked phosphatase activity. Our results indicate that the diverse phenotypes observed in Inpp4a-related neurological diseases could be due to the varying protein expression levels and retained phosphatase activity in different Inpp4a variants. These findings provide insights into the role of INPP4A mutations in disease pathogenesis and may help to develop personalized therapy.

INPP5D modulates TREM2 loss-of-function phenotypes in a ß-amyloidosis mouse model.

The genetic associations of TREM2 loss-of-function variants with Alzheimer disease (AD) indicate the protective roles of microglia in AD pathogenesis. Functional deficiencies of TREM2 disrupt microglial clustering around amyloid ß (Aß) plaques, impair their transcriptional response to Aß, and worsen neuritic dystrophy. However, the molecular mechanism underlying these phenotypes remains unclear. In this study, we investigated the pathological role of another AD risk gene, INPP5D, encoding a phosphoinositide PI(3,4,5)P3 phosphatase expressed in microglia. In a Tyrobp-deficient TREM2 loss-of-function mouse model, Inpp5d haplodeficiency restored the association of microglia with Aß plaques, partially restored plaque compaction, and astrogliosis, and reduced phosphorylated tau+ dystrophic neurites. Mechanistic analyses suggest that TREM2/TYROBP and INPP5D exert opposing effects on PI(3,4,5)P3 signaling pathways as well as on phosphoproteins involved in the actin assembly. Our results suggest that INPP5D acts downstream of TREM2/TYROBP to regulate the microglial barrier against Aß toxicity, thereby modulates Aß-dependent pathological conversion of tau.

N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine inhibits bladder cancer progression by suppressing YAP1/TAZ.

Bladder cancer (BlC) is the fourth most common cancer in males worldwide, but few systemic chemotherapy options for its effective treatment exist. The development of new molecularly-targeted agents against BlC is therefore an urgent issue. The Hippo signaling pathway, with its upstream LATS kinases and downstream transcriptional co-activators YAP1 and TAZ, plays a pivotal role in diverse cell functions, including cell proliferation. Recent studies have shown that overexpression of YAP1 occurs in advanced BlCs and is associated with poor patient prognosis. Accessing data from our previous screening of a chemical library of compounds targeting the Hippo pathway, we identified DMPCA (N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine) as an agent able to induce the phosphorylation of LATS1 and YAP1/TAZ in BlC cells, thereby suppressing their viability both in vitro and in mouse xenografts. Our data indicate that DMPCA has a potent anti-tumor effect, and raise the possibility that this agent may represent a new and effective therapeutic option for BlC.

Plasma membrane phosphatidylinositol (4,5)-bisphosphate is critical for determination of epithelial characteristics.

Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.

A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation.

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.

PI4P/PS countertransport by ORP10 at ER-endosome membrane contact sites regulates endosome fission.

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER-endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER-endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER-endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.

Altering phosphoinositides in high-fat diet-associated prostate tumor xenograft growth.

The metabolic reprogramming of phospholipids may affect intracellular signal transduction pathways. A high-fat diet (HFD) is attributed to prostate cancer (PCa) progression, but the expression pattern and role of phospholipids in HFD-mediated PCa progression remains unclear. In this study, HFD enhanced LNCaP xenograft tumor growth by upregulating the phosphatidylinositol (PI) 3-kinase (PI3K)/AKT signaling pathway. A lipidomic analysis using xenograft tumors showed that phosphoinositides, especially PI (3,4,5)-trisphosphate (PIP3), including several species containing C38:4, C38:3, and C40:4 fatty acids, increased in the HFD group compared to control. Fatty acid synthase (FASN) was significantly upregulated in xenograft tumors under HFD in both gene and protein levels. PCa cell growth was significantly inhibited through the decreased AKT signaling pathway by treatment with cerulenin, a chemical FASN inhibitor, which also downregulated PIP, PIP2, and PIP3 but not PI. Thus, dietary fat influences PCa progression and alters phosphoinositides, especially PIP3, a critical player in the PI3K/AKT pathway. These results may offer appropriate targets, such as FASN, for dietary intervention and/or chemoprevention to reduce PCa incidence and progression.

Endogenous YAP1 activation drives immediate onset of cervical carcinoma in situ in mice.

Cervical cancer (CC) is usually initiated by infection with high-risk types of human papillomavirus (HPV). The HPV E6 and E7 proteins target p53 and RB, respectively, but other cellular targets likely exist. We generated uterus-specific MOB1A/B double KO (uMob1DKO) mice, which immediately developed cervical squamous cell carcinoma in situ. Mutant cervical epithelial cells showed YAP1-dependent hyperproliferation, altered self-renewal, impaired contact inhibition, and chromosomal instability. p53 activation was increased in uMob1DKO cells, and additional p53 loss in uMob1DKO mice accelerated tumor invasion. In human CC, strong YAP1 activation was observed from the precancerous stage. Human cells overexpressing HPV16 E6/E7 showed inactivation of not only p53 and RB but also PTPN14, boosting YAP1 activation. Estrogen, cigarette smoke condensate, and PI3K hyperactivation all increased YAP1 activity in human cervical epithelial cells, and PTPN14 depletion along with PI3K activation or estrogen treatment further enhanced YAP1. Thus, immediate CC onset may initiate when YAP1 activity exceeds an oncogenic threshold, making Hippo-YAP1 signaling a major CC driver.

Increased fatty acyl saturation of phosphatidylinositol phosphates in prostate cancer progression.

Phosphoinositides (PIPs) participate in many cellular processes, including cancer progression; however, the metabolic features of PIPs associated with prostate cancer (PCa) are unknown. We investigated PIPs profiles in PTEN-deficient prostate cancer cell lines, human prostate tissues obtained from patients with PCa and benign prostate hyperplasia (BPH) specimens using mass spectrometry. In immortalized normal human prostate PNT1B cells, PTEN deficiency increased phosphatidylinositol tris-phosphate (PIP3) and decreased phosphatidylinositol mono- and bis-phosphate (PIP1 and PIP2), consistent with PTEN's functional role as a PI(3,4,5)P3 3-phosphatase. In human prostate tissues, levels of total (sum of all acyl variants) phosphatidylinositol (PI) and PIP1 in PCa were significantly higher than in BPH, whereas PIP2 and PIP3 contents were significantly lower than in BPH. PCa patients had significantly higher proportion of PI, PIP1, and PIP2 with 0-2 double bonds in acyl chains than BPH patients. In subgroup analyses based on PCa aggressiveness, mean total levels of PI with 0-2 double bonds in acyl chains were significantly higher in patients with pathological stage T3 than in those with pathological stage T2. These data indicate that alteration of PIPs level and the saturation of acyl chains may be associated with the development and aggressiveness of prostate cancer, although it is unknown whether this alteration is causative.

NH3 -Driven Benzene C-H Activation with O2 that Opens a New Way for Selective Phenol Synthesis.

Catalytic benzene C-H activation toward selective phenol synthesis with O2 remains a stimulating challenge to be tackled. Phenol is currently produced industrially by the three-steps cumene process in liquid phase, which is energy-intensive and not environmentally friendly. Hence, there is a strong demand for an alternative gas-phase single-path reaction process. This account documents the pivotal confined single metal ion site platform with a sufficiently large coordination sphere in ß zeolite pores, which promotes the unprecedented catalysis for the selective benzene hydroxylation with O2 under coexisting NH3 by the new inter-ligand concerted mechanism. Among alkali and alkaline-earth metal ions and transition and precious metal ions, single Cs+ and Rb+ sites with ion diameters >0.300 nm in the ß pores exhibited good performances for the direct phenol synthesis in a gas-phase single-path reaction process. The single Cs+ and Rb+ sites that possess neither significant Lewis acidic-basic property nor redox property, cannot activate benzene, O2 , and NH3 , respectively, whereas when they coadsorbed together, the reaction of the inter-coadsorbates on the single alkali-metal ion site proceeds concertedly (the inter-ligand concerted mechanism), bringing about the benzene C-H activation toward phenol synthesis. The NH3 -driven benzene C-H activation with O2 was compared to the switchover of the reaction pathways from the deep oxidation to selective oxidation of benzene by coexisting NH3 on Pt6 metallic cluster/ß and Ni4 O4 oxide cluster/ß. The NH3 -driven selective oxidation mechanism observed with the Cs+ /ß and Rb+ /ß differs from the traditional redox catalysis (Mars-van Krevelen) mechanism, simple Langmuir-Hinshelwood mechanism, and acid-base catalysis mechanism involving clearly defined interaction modes. The present catalysis concept opens a new way for catalytic selective oxidation processes involving direct phenol synthesis.

Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

Cell competition is involved in mammalian embryogenesis and tumor elimination and progression. It was previously shown that, whereas NIH3T3 mouse fibroblasts expressing high levels of the yes-associated protein 1(YAP1) target TEA domain family (TEAD) transcription factors become "winners" in cell competitions, Madin-Darby canine kidney cells expressing activated YAP1 become "losers" and are eliminated from culture monolayers. Thus, YAP1's role in cell competitions is clearly context dependent. Here, we show that keratinocytes overexpressing a constitutively activated YAP1 mutant lose in in vitro competitions with control cells conducted in standard tissue culture dishes and undergo apical extrusion. Similarly, cells in which endogenous YAP1 is activated by NF2 knockdown become losers. The YAP1-overexpressing cells exhibit a decrease in cell-matrix adhesion because of defective expression of adhesion molecules such as fibronectin-1. Cell adhesion-mediated proliferation is also impaired. However, because of intrinsic factors, YAP1-expressing cells proliferate faster than control cells when cocultured in dishes impeding cell adhesion. In vivo, Mob1a/b-deficient (YAP1-activated) epidermis, which shows decreased expression of type XVII collagen, cannot be engrafted successfully onto donor mice. YAP1-activated skin grafts shrink away from surrounding control skin, and the epidermis peels off the basement membrane. Our data show that YAP1 activation controls cell competition in part by decreasing cell adhesion.-Nishio, M., Miyachi, Y., Otani, J., Tane, S., Omori, H., Ueda, F., Togashi, H., Sasaki, T., Mak, T. W., Nakao, K., Fujita, Y., Nishina, H., Maehama, T., Suzuki, A. Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

Metabolic Determinants of Sensitivity to Phosphatidylinositol 3-Kinase Pathway Inhibitor in Small-Cell Lung Carcinoma.

Comprehensive genomic analysis has revealed that the PI3K/AKT/mTOR pathway is a feasible therapeutic target in small-cell lung carcinoma (SCLC). However, biomarkers to identify patients likely to benefit from inhibitors of this pathway have not been identified. Here, we show that metabolic features determine sensitivity to the PI3K/mTOR dual inhibitor gedatolisib in SCLC cells. Substantial phosphatidyl lipid analysis revealed that a specific phosphatidylinositol (3,4,5)-trisphosphate (PIP3) subspecies lipid product PIP3 (38:4) is predictive in assessing sensitivity to PI3K/mTOR dual inhibitor. Notably, we found that higher amounts of purine-related aqueous metabolites such as hypoxanthine, which are characteristic of SCLC biology, lead to resistance to PI3K pathway inhibition. In addition, the levels of the mRNA encoding hypoxanthine phosphoribosyl transferase 1, a key component of the purine salvage pathway, differed significantly between SCLC cells sensitive or resistant to gedatolisib. Moreover, complementation with purine metabolites could reverse the vulnerability to targeting of the PI3K pathway in SCLC cells normally sensitive to gedatolisib. These results indicate that the resistance mechanism of PI3K pathway inhibitors is mediated by the activation of the purine salvage pathway, supplying purine resource to nucleotide biosynthesis. Metabolomics is a powerful approach for finding novel therapeutic biomarkers in SCLC treatment.Significance: These findings identify features that determine sensitivity of SCLC to PI3K pathway inhibition and support metabolomics as a tool for finding novel therapeutic biomarkers. Cancer Res; 78(9); 2179-90. ©2018 AACR.

TMEM55a localizes to macrophage phagosomes to downregulate phagocytosis.

TMEM55a (also known as PIP4P2) is an enzyme that dephosphorylates the phosphatidylinositol (PtdIns) PtdIns(4,5)P2 to form PtdIns(5)P in vitro However, the in vivo conversion of the polyphosphoinositide into PtdIns(5)P by the phosphatase has not yet been demonstrated, and the role of TMEM55a remains poorly understood. Here, we found that mouse macrophages (Raw264.7) deficient in TMEM55a showed an increased engulfment of large particles without affecting the phagocytosis of Escherichia coli Transfection of a bacterial phosphatase with similar substrate specificity to TMEM55a, namely IpgD, into Raw264.7 cells inhibited the engulfment of IgG-erythrocytes in a manner dependent on its phosphatase activity. In contrast, cells transfected with PIP4K2a, which catalyzes PtdIns(4,5)P2 production from PtdIns(5)P, increased phagocytosis. Fluorescent TMEM55a transfected into Raw264.7 cells was found to mostly localize to the phagosome. The accumulation of PtdIns(4,5)P2, PtdIns(3,4,5)P3 and F-actin on the phagocytic cup was increased in TMEM55a-deficient cells, as monitored by live-cell imaging. Phagosomal PtdIns(5)P was decreased in the knockdown cells, but the augmentation of phagocytosis in these cells was unaffected by the exogenous addition of PtdIns(5)P. Taken together, these results suggest that TMEM55a negatively regulates the phagocytosis of large particles by reducing phagosomal PtdIns(4,5)P2 accumulation during cup formation.

PTEN Regulates PI(3,4)P2 Signaling Downstream of Class I PI3K.

The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.

Fatty acid binding protein 4 enhances prostate cancer progression by upregulating matrix metalloproteinases and stromal cell cytokine production.

Fatty acid binding protein 4 (FABP4) is an abundant protein in adipocytes, and its production is influenced by high-fat diet (HFD) or obesity. The prostate stromal microenvironment induces proinflammatory cytokine production, which is key for the development and progression of prostate cancer (PCa). Here, we show that high FABP4 expression and its secretion by PCa cells directly stimulated PCa cell invasiveness by upregulating matrix metalloproteinases through phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways. In addition, prostate stromal cells augmented PCa cell invasiveness by secreting interleukin-8 and -6 in response to FABP4. This was abrogated by the FABP4 specific inhibitor, BMS309403. Furthermore, a mouse xenograft experiment showed HFD enhanced PCa metastasis and invasiveness by the upregulation of FABP4 and interleukin-8. Clinically, the serum level of FABP4 was significantly associated with an aggressive type of PCa rather than obesity. Taken together, FABP4 may enhance PCa progression and invasiveness by upregulating matrix metalloproteinases and cytokine production in the PCa stromal microenvironment, especially under HFD or obesity.

Myeloid cell-specific inositol polyphosphate-4-phosphatase type I knockout mice impair bacteria clearance in a murine peritonitis model.

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling has been implicated in the anti-inflammatory response in a mouse model of endotoxemia and sepsis. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a), which dephosphorylates PtdIns(3,4)P2 to PtdIns(3)P, in bacterial infections. We prepared myeloid cell-specific Inpp4a-conditional knockout mice. Macrophages from these mice showed increased Akt phosphorylation and reduced production of inflammatory cytokines in response to LPS or Escherichia coli in vitro The Inpp4a knockout mice survived for a shorter time than wild type mice after i.p. infection with E. coli, with less production of inflammatory cytokines. Additionally, E. coli clearance from blood and lung was significantly impaired in the knockout mice. A likely mechanism is that the Inpp4a-catalyzed dephosphorylation of PtdIns(3,4)P2 down-regulates Akt pathways, which, in turn, increases the production of inflammatory mediators. This mechanism at least fits the decreased E. coli clearance and short survival in the Inpp4a knockout mice.