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Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of [p47.sup.phox], a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of [p47.sup.phox] with phosphoinositides. Although the isolated phox homology (PX) domain of [p47.sup.phox] can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length [p47.sup.phox] is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of [p47.sup.phox] to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of [p47.sup.phox], because protein kinase C treatment of the wild-type [p47.sup.phox] but not of a mutant protein with the S303/ 304/328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type [p47.sup.phox] translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive [p47.sup.phox] nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of [p47.sup.phox] enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of [p47.sup.phox] from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.

Background: Migration of cancer cell correlates with distant metastasis and local invasion, which are good targets for cancer treatment. An optically accessible device "TAXIScan" was developed, which provides considerably more information regarding the cellular dynamics and less quantity of samples than do the existing methods. Here, we report the establishment of a system to analyze the nature of pancreatic cancer cells using TAXIScan and we evaluated lysophosphatidic acid (LPA)-elicited pancreatic cell migration.Methods: Pancreatic cancer cell lines, BxPC3, PANC-1, AsPC1, and MIAPaCa-2, were analyzed for adhesion as well as migration towards LPA by TAXIScan using parameters such as velocity and directionality or for the number of migrated cells by the Boyden chamber methods. To confirm that the migration was initiated by LPA, the expression of LPA receptors and activation of intracellular signal transductions were examined by quantitative reverse transcriptase polymerase reaction and western blotting.Results: Scaffold coating was necessary for the adhesion of pancreatic cancer cells, and collagen I and Matrigel were found to be good scaffolds. BxPC3 and PANC-1 cells clearly migrated towards the concentration gradient formed by injecting 1 μL LPA, which was abrogated by pre-treatment with LPA inhibitor, Ki16425 (IC50 for the directionality ≈ 1.86 μM). The LPA dependent migration was further confirmed by mRNA and protein expression of LPA receptors as well as phosphorylation of signaling molecules. LPA1 mRNA was highest among the 6 receptors, and LPA1, LPA2 and LPA3 proteins were detected in BxPC3 and PANC-1 cells. Phosphorylation of Akt (Thr308 and Ser473) and p42/44MAPK in BxPC3 and PANC-1 cells was observed after LPA stimulation, which was clearly inhibited by pre-treatment with a compound Ki16425.Conclusions: We established a novel pancreatic cancer cell migration assay system using TAXIScan. This assay device provides multiple information on migrating cells simultaneously, such as their morphology, directionality, and velocity, with a small volume of sample and can be a powerful tool for analyzing the nature of cancer cells and for identifying new factors that affect cell functions. [ABSTRACT FROM AUTHOR]

The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacteriumAnaplasma phagocytophiluminhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated.bfl-1mRNA levels in uninfected neutrophils after 12 h in culture were reduced to≈ 5–25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance ofbfl-1mRNA levels byA. phagocytophilum. Differences inmcl-1,bax,bcl-w,badorbakmRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10–12 h incubation, whereasA. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner inA. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest thatA. phagocytophiluminhibits human neutrophil apoptosis via transcriptional upregulation ofbfl-1and inhibition of mitochondria-mediated activation of caspase 3. [ABSTRACT FROM AUTHOR]

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67phox and p47phox. They translocate to the membrane upon cell stimulation and activate flavocytochrome b558, the membrane-integrated catalytic core of this enzyme system. The activators p67phox and p47phox form a ternary complex together with p40phox, an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif-containing domains: p40phox associates with p67phox via binding of the p40phox PC motif to the p67phox PB1 domain, while p47phox directly interacts with p67phox but not with p40phox. Here we show that p40phox enhances membrane translocation of p67phox and p47phox in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40phox carrying the D289A substitution in PC or a p67phox with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40phox participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67phox and p47phox via the PB1-PC interaction with p67phox. [ABSTRACT FROM AUTHOR]

Background: EMT has been proposed to be a crucial early event in cancer metastasis. EMT is rigidly regulated by the action of several EMT-core transcription factors, particularly ZEB1. We previously revealed an unusual role of ZEB1 in the S100A8/A9-mediated metastasis in breast cancer cells that expressed ZEB1 at a significant level and showed that the ZEB1 was activated on the MCAM-downstream pathway upon S100A8/A9 binding. ZEB1 is well known to require Zn 2+ for its activation based on the presence of several Zn-finger motifs in the transcription factor. However, how Zn 2+ -binding works on the pleiotropic role of ZEB1 through cancer progression has not been fully elucidated., Methods: We established the engineered cells, MDA-MB-231 MutZEB1 (MDA-MutZEB1), that stably express MutZEB1 (ΔZn). The cells were then evaluated in vitro for their invasion activities. Finally, an RNA-Seq analysis was performed to compare the gene alteration profiles of the established cells comprehensively., Results: MDA-MutZEB1 showed a significant loss of the EMT, ultimately stalling the invasion. Inclusive analysis of the transcription changes after the expression of MutZEB1 (ΔZn) in MDA-MB-231 cells revealed the significant downregulation of LOX family genes, which are known to play a critical role in cancer metastasis. We found that LOXL1 and LOXL4 remarkably enhanced cancer invasiveness among the LOX family genes with altered expression., Conclusions: These findings indicate that ZEB1 potentiates Zn 2+ -mediated transcription of plural EMT-relevant factors, including LOXL1 and LOXL4, whose upregulation plays a critical role in the invasive dissemination of breast cancer cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hirabayashi, Yamamoto, Maruyama, Tomonobu, Kinoshita, Chen, Komalasari, Murata, Gohara, Jiang, Zhou, Ruma, Sumardika, Yamauchi, Kuribayashi, Toyooka, Inoue and Sakaguchi.)

Background & Aims: CD26, a multifunctional transmembrane glycoprotein, is expressed in various cancers and functions as dipeptidyl peptidase 4 (DPP4). We investigated whether CD26 expression is associated with hepatocellular carcinoma (HCC) progression and whether DPP4 inhibitors exert antitumor effects against HCC., Methods: CD26 expression was examined in 41 surgically resected HCC specimens. The effects of DPP4 inhibitors on HCC were examined by using HCC cell lines (Huh-7 and Li-7), xenograft tumors in nude mice, and a nonalcoholic steatohepatitis-related HCC mouse model., Results: CD26 expression in HCC specimens was associated with increased serum DPP4 activity, as well as a more advanced stage, less tumor immunity, and poorer prognosis in HCC patients. The HCC cell lines and xenograft tumors exhibited CD26 expression and DPP4 activity. The DPP4 inhibitors did not exhibit antitumor effects in vitro, but natural killer (NK) and/or T-cell tumor accumulation suppressed growth of xenograft tumor and HCC in vivo. The antitumor effects of DPP4 inhibitors were abolished by the depletion of NK cells or the neutralization of CXCR3, a chemokine receptor on NK cells. EZ-TAXIScan, an optical horizontal chemotaxis apparatus, identified enhanced NK and T-cell chemotaxis by DPP4 inhibitors ex vivo in the presence of Huh-7 cells and the chemokine CXCL10, which binds to CXCR3. The DPP4 inhibitors prevented the biologically active form of CXCL10 from being truncated by Huh-7 cell DPP4 activity. DPP4 inhibitors also suppressed tumor angiogenesis., Conclusions: These results provide a rationale for verifying whether DPP4 inhibitors clinically inhibit the progression of HCC or augment the antitumor effects of molecular-targeting drugs or immunotherapies against HCC.