Endosomes In Cells Research Articles

Anti-IgE Qb-VLP Conjugate Vaccine Self-Adjuvants through Activation of TLR7.

Qb bacteriophage virus-like particles (Qb-VLP) are utilized as carriers to enhance immune responses to weakly or non-immunogenic antigens such as peptides and haptens. Qb-VLPs are formed through the self-assembly of multiple Qb capsid protein monomers, a process which traps a large amount of bacterial RNA in the core of the VLP. Bacterial RNA is known to activate the innate immune system via TLR 7 and 8 found within the endosomes of certain immune cells and has been shown to contribute to the immunogenicity of Qb-VLP vaccines. Herein, we evaluated an anti-IgE vaccine comprised of two IgE peptides (Y and P) conjugated to Qb-VLP (Qb-Y and Qb-P, respectively) for in vitro stimulation of human PBMCs and in vivo immunogenicity in mice. The in vitro secretion of IFN-α from human PBMCs exposed to Qb-Y is consistent with TLR7 activation. Immunization of mice with the IgE peptide Qb-VLP conjugates induced high titers of anti-IgE antibodies in wild-type mice, but significantly lower titers in TLR7 knockout mice, supporting the self-adjuvanting role of the RNA. Inclusion of alum and alum/CpG as adjuvants partially or completely compensated for the lack of TLR7 activation in TLR7-deficient mice. Our study demonstrates the key role that TLR7 plays in the immunogenicity of the IgE peptide Qb-VLP conjugate vaccine.

Grancalcin (GCA) modulates Toll‐like receptor 9 (TLR9) mediated signaling through its direct interaction with TLR9

Toll-like receptors (TLRs) are playing important roles in stimulating the innate immune response and intensifying adaptive immune response against invading pathogens. Appropriate regulation of TLR activation is important to maintain a balance between preventing tumor activation and inhibiting autoimmunity. Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells and triggers myeloid differentiation primary response gene 88 (MyD88) dependent nuclear factor kappa B (NF-κB) pathways and type I interferon (IFN) responses. However, mechanisms of how TLR9 signals are mediated and which molecules are involved in controlling TLR9 functions remain poorly understood. Here, we report that penta EF-hand protein grancalcin (GCA) interacts and binds with TLR9 in a yeast two-hybrid system and an overexpression system. Using siRNA-mediated knockdown experiments, we also revealed that GCA positively regulates type I IFN production, cytokine/chemokine production through nuclear localization of interferon regulatory factor 7 (IRF7), NF-κB activation, and mitogen-activated protein kinase (MAPK) activation in plasmacytoid dendritic cells. Our results indicate that heterodimerization of GCA and TLR9 is important for TLR9-mediated downstream signaling and might serve to fine tune processes against viral infection.

Protocols for Exosome Isolation and RNA Profiling.

Exosomes are small extracellular vesicles of multivesicular bodies derived from the cell endosome. Molecules have a characteristic composition both in the membrane and in the carried load. These particles are located in the culture medium of cells in vitro and in vivo body fluids, being synthesized by almost all kind of cells. The most characteristic molecule transported within is the RNA with a large clinical potential. In this chapter, the methodology for isolation of exosomes and subsequent purification of the RNA is reviewed.

Zirconium Phosphate Nanoplatelet Potential for Anticancer Drug Delivery Applications.

Zirconium phosphate (ZrP) nanoplatelets can intercalate anticancer agents via an ion exchange reaction creating an inorganic delivery system with potential for cancer treatment. ZrP delivery of anticancer agents inside tumor cells was explored in vitro. Internalization and cytotoxicity of ZrP nanoplatelets were studied in MCF-7 and MCF-10A cells. DOX-loaded ZrP nanoplatelets (DOX@ZrP) uptake was assessed by confocal (CLSM) and transmission electron microscopy (TEM). Cytotoxicity to MCF-7 and MCF-10A cells was determined by the MTT assay. Reactive Oxy- gen Species (ROS) production was analyzed by fluorometric assay, and cell cycle alterations and induction of apoptosis were analyzed by flow cytometry. ZrP nanoplatelets were localized in the endosomes of MCF-7 cells. DOX and ZrP nanoplatelets were co-internalized into MCF-7 cells as detected by CLSM. While ZrP showed limited toxicity to MCF-7 cells, DOX@ZrP was cytotoxic at an IC₅₀ similar to that of free DOX. Meanwhile, DOX lC₅₀ was significantly lower than the equivalent concentration of DOX@ZrP in MCF-10A cells. ZrP did not induce apoptosis in both cell lines. DOX and DOX@ZrP induced significant oxidative stress in both cell models. Results suggest that ZrP nanoplatelets are promising as carriers of anticancer agents into cancer cells.

Bidirectional traffic between the Golgi and the endosomes - machineries and regulation.

The bidirectional transport between the Golgi complex and the endocytic pathway has to be finely regulated in order to ensure the proper delivery of newly synthetized lysosomal enzymes and the return of sorting receptors from degradative compartments. The high complexity of these routes has led to experimental difficulties in properly dissecting and separating the different pathways. As a consequence, several models have been proposed during the past decades. However, recent advances in our understanding of endosomal dynamics have helped to unify these different views. We provide here an overview of the current insights into the transport routes between Golgi and endosomes in mammalian cells. The focus of the Commentary is on the key molecules involved in the trafficking pathways between these intracellular compartments, such as Rab proteins and sorting receptors, and their regulation. A proper understanding of the bidirectional traffic between the Golgi complex and the endolysosomal system is of uttermost importance, as several studies have demonstrated that mutations in the factors involved in these transport pathways result in various pathologies, in particular lysosome-associated diseases and diverse neurological disorders, such as Alzheimer's and Parkinson's disease.

The Mammalian Orthologs of Drosophila Lgd, CC2D1A and CC2D1B, Function in the Endocytic Pathway, but Their Individual Loss of Function Does Not Affect Notch Signalling.

CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.

Novel Role of TRPML2 in the Regulation of the Innate Immune Response

TRPMLs (or mucolipins) constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the TRPML family includes three members: TRPML1-3. Although TRPML1 and TRPML3 have been well characterized, the cellular function of TRPML2 has remained elusive. To address TRPML2 function in a physiologically relevant cell type, we first analyzed TRPML2 expression in different mouse tissues and organs and found that it was predominantly expressed in lymphoid organs and kidney. Quantitative RT-PCR revealed tight regulation of TRPML2 at the transcriptional level. Although TRPML2 expression was negligible in resting macrophages, TRPML2 mRNA and protein levels dramatically increased in response to TLR activation both in vitro and in vivo. Conversely, TRPML1 and TRPML3 levels did not change upon TLR activation. Immunofluorescence analysis demonstrated that endogenous TRPML2 primarily localized to recycling endosomes both in culture and primary cells, in contrast with TRPML1 and TRPML3, which distribute to the late and early endosomal pathway, respectively. To better understand the in vivo function of TRPML2, we generated a TRPML2-knockout mouse. We found that the production of several chemokines, in particular CCL2, was severely reduced in TRPML2-knockout mice. Furthermore, TRPML2-knockout mice displayed impaired recruitment of peripheral macrophages in response to i.p. injections of LPS or live bacteria, suggesting a potential defect in the immune response. Overall, our study reveals interesting differences in the regulation and distribution of the members of the TRPML family and identifies a novel role for TRPML2 in the innate immune response.

Distinct Trafficking of Cell Surface and Endosomal TIM‐1 to the Immune Synapse

The T cell costimulatory molecule TIM-1 (T cell/transmembrane, mucin and immunoglobulin domain protein 1) sorts mainly to endosomes in lymphoid cells. At difference from the cell surface protein, endosomal TIM-1 translocates to the immune synapse (IS), where it can contribute to antigen-dependent T cell costimulation. TIM-1 ligands increase the amount of cell surface protein, preventing its traffic to the IS. The bipolar sorting of TIM-1 observed during IS formation is determined by differences in its subcellular location, and probably modulates antigen-driven immune responses.

Influence of the proton pump inhibitor lansoprazole on distribution and activity of doxorubicin in solid tumors.

Cellular causes of resistance and limited drug distribution within solid tumors limit therapeutic efficacy of anticancer drugs. Acidic endosomes in cancer cells mediate autophagy, which facilitates survival of stressed cells, and may contribute to drug resistance. Basic drugs (e.g. doxorubicin) are sequestered in acidic endosomes, thereby diverting drugs from their target DNA and decreasing penetration to distal cells. Proton pump inhibitors (PPIs) may raise endosomal pH, with potential to improve drug efficacy and distribution in solid tumors. We determined the effects of the PPI lansoprazole to modify the activity of doxorubicin. To gain insight into its mechanisms, we studied the effects of lansoprazole on endosomal pH, and on the spatial distribution of doxorubicin, and of biomarkers reflecting its activity, using in vitro and murine models. Lansoprazole showed concentration-dependent effects to raise endosomal pH and to inhibit endosomal sequestration of doxorubicin in cultured tumor cells. Lansoprazole was not toxic to cancer cells but potentiated the cytotoxicity of doxorubicin and enhanced its penetration through multilayered cell cultures. In solid tumors, lansoprazole improved the distribution of doxorubicin but also increased expression of biomarkers of drug activity throughout the tumor. Combined treatment with lansoprazole and doxorubicin was more effective in delaying tumor growth as compared to either agent alone. Together, lansoprazole enhances the therapeutic effects of doxorubicin both by improving its distribution and increasing its activity in solid tumors. Use of PPIs to improve drug distribution and to inhibit autophagy represents a promising strategy to enhance the effectiveness of anticancer drugs in solid tumors.

Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells

Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

In Vivo Role of INPP4B in Tumor and Metastasis Suppression through Regulation of PI3K-AKT Signaling at Endosomes.

The phosphatases PTEN and INPP4B have been proposed to act as tumor suppressors by antagonizing PI3K-AKT signaling and are frequently dysregulated in human cancer. Although PTEN has been extensively studied, little is known about the underlying mechanisms by which INPP4B exerts its tumor-suppressive function and its role in tumorigenesis in vivo. Here, we show that a partial or complete loss of Inpp4b morphs benign thyroid adenoma lesions in Pten heterozygous mice into lethal and metastatic follicular-like thyroid cancer (FTC). Importantly, analyses of human thyroid cancer cell lines and specimens reveal INPP4B downregulation in FTC. Mechanistically, we find that INPP4B, but not PTEN, is enriched in the early endosomes of thyroid cancer cells, where it selectively inhibits AKT2 activation and in turn tumor proliferation and anchorage-independent growth. We therefore identify INPP4B as a novel tumor suppressor in FTC oncogenesis and metastasis through localized regulation of the PI3K-AKT pathway at the endosomes. Although both PTEN and INPP4B can inhibit PI3K-AKT signaling through their lipid phosphatase activities, here we demonstrate lack of an epistatic relationship between the two tumor suppressors. Instead, the qualitative regulation of PI3K-AKT2 signaling by INPP4B provides a mechanism for their cooperation in suppressing thyroid tumorigenesis and metastasis.

Cathepsin W Is Required for Escape of Influenza A Virus from Late Endosomes.

ABSTRACTHuman cathepsin W (CtsW) is a cysteine protease, which was identified in a genome-wide RNA interference (RNAi) screen to be required for influenza A virus (IAV) replication. In this study, we show that reducing the levels of expression of CtsW reduces viral titers for different subtypes of IAV, and we map the target step of CtsW requirement to viral entry. Using a set of small interfering RNAs (siRNAs) targeting CtsW, we demonstrate that knockdown of CtsW results in a decrease of IAV nucleoprotein accumulation in the nuclei of infected cells at 3 h postinfection. Assays specific for the individual stages of IAV entry further show that attachment, internalization, and early endosomal trafficking are not affected by CtsW knockdown. However, we detected impaired escape of viral particles from late endosomes in CtsW knockdown cells. Moreover, fusion analysis with a dual-labeled influenza virus revealed a significant reduction in fusion events, with no detectable impact on endosomal pH, suggesting that CtsW is required at the stage of viral fusion. The defect in IAV entry upon CtsW knockdown could be rescued by ectopic expression of wild-type CtsW but not by the expression of a catalytically inactive mutant of CtsW, suggesting that the proteolytic activity of CtsW is required for successful entry of IAV. Our results establish CtsW as an important host factor for entry of IAV into target cells and suggest that CtsW could be a promising target for the development of future antiviral drugs.

Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology.

Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail.

ER-endosome contact sites: molecular compositions and functions.

Recent studies have revealed the existence of numerous contact sites between the endoplasmic reticulum (ER) and endosomes in mammalian cells. Such contacts increase during endosome maturation and play key roles in cholesterol transfer, endosome positioning, receptor dephosphorylation, and endosome fission. At least 7 distinct contact sites between the ER and endosomes have been identified to date, which have diverse molecular compositions. Common to these contact sites is that they impose a close apposition between the ER and endosome membranes, which excludes membrane fusion while allowing the flow of molecular signals between the two membranes, in the form of enzymatic modifications, or ion, lipid, or protein transfer. Thus, ER-endosome contact sites ensure coordination of molecular activities between the two compartments while keeping their general compositions intact. Here, we review the molecular architectures and cellular functions of known ER-endosome contact sites and discuss their implications for human health.

UDP-D-galactose synthesis by UDP-glucose 4-epimerase 4 is required for organization of the trans-Golgi network/early endosome in Arabidopsis thaliana root epidermal cells.

Endomembrane organization is essential for cell physiology. We previously identified an Arabidopsis thaliana mutant in which a plasma membrane (PM) marker GFP-NIP5;1 and trans-Golgi network/early endosome (TGN/EE) markers were accumulated in intracellular aggregates in epidermal cells of the root elongation zone. The mutant was identified as an allele of UDP-glucose epimerase 4 (UGE4)/root hair defective 1/root epidermal bulgar 1, which was previously described as a mutant with swollen root epidermal cells and has an altered sugar composition in cell wall polysaccharides. Importantly, these defects including aggregate formation were restored by supplementation of D-galactose in the medium. These results suggested that UDP-D-galactose synthesis by UGE4 is important for endomembrane organization in addition to cell wall structure. Here, we further investigated the nature of the aggregates using various markers of endomembrane compartments and BOR1-GFP, which traffics from PM to vacuole in response to high-B supply. The markers of multi-vesicular bodies/late endosomes (MVB/LEs) and BOR1-GFP were strongly accumulated in the intracellular aggregates, while those of the endoplasmic reticulum (ER), the vacuolar membrane, and the Golgi were only slightly affected in the uge4 mutant. The abnormal localizations of these markers in the uge4 mutant differed from the effects of inhibitors of actin and microtubule polymerization, although they also affected endomembrane organization. Furthermore, electron microscopy analysis revealed accumulation of abnormal high-electron-density vesicles in elongating epidermal cells. The abnormal vesicles were often associated or interconnected with TGN/EEs and contained ADP-ribosylation factor 1, which is usually localized to the Golgi and the TGN/EEs. On the other hand, structures of the ER, Golgi apparatus, and MVB/LEs were apparently normal in uge4 cells. Together, our data indicate the importance of UDP-D-galactose synthesis by UGE4 for the organization and function of endomembranes, especially TGN/EEs, which are a sorting station of the secretory and vacuolar pathways.

Chloride transporters and receptor-mediated endocytosis in the renal proximal tubule.

The reabsorptive activity of renal proximal tubule cells is mediated by receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Loss-of-function mutations of the endosomal chloride-proton exchanger ClC-5 (Dent's disease) cause a major trafficking defect in proximal tubule cells, associated with lysosomal dysfunction, oxidative stress and dedifferentiation/proliferation. A similar but milder defect is associated with mutations in CFTR (cystic fibrosis transmembrane conductance regulator). Vesicular chloride transport appears to be important for the integrity of the endolysosomal pathway in epithelial cells. The epithelial cells lining the proximal tubules of the kidney reabsorb a large amount of filtered ions and solutes owing to receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Dedifferentiation of proximal tubule cells and dysfunction of receptor-mediated endocytosis characterize Dent's disease, a rare disorder caused by inactivating mutations in the CLCN5 gene that encodes the endosomal chloride-proton exchanger, ClC-5. The disease is characterized by a massive urinary loss of solutes (renal Fanconi syndrome), with severe metabolic complications and progressive renal failure. Investigations of mutations affecting the gating of ClC-5 revealed that the proximal tubule dysfunction may occur despite normal endosomal acidification. In addition to defective endocytosis, proximal tubule cells lacking ClC-5 show a trafficking defect in apical receptors and transporters, as well as lysosomal dysfunction and typical features of dedifferentiation, proliferation and oxidative stress. A similar but milder defect is observed in mouse models with defective CFTR, a chloride channel that is also expressed in the endosomes of proximal tubule cells. These data suggest a major role for endosomal chloride transport in the maintenance of epithelial differentiation and reabsorption capacity of the renal proximal tubule.

PH-triggered release of manganese from MnAu nanoparticles that enables cellular neuronal differentiation without cellular toxicity

At high concentrations, manganese (Mn) promotes cellular neurodevelopment but causes toxicity. Here, we report that Mn ion at high concentrations can be delivered to pheochromocytoma 12 (PC12) cells using gold nanoparticles (AuNPs) to enhance cellular neurodevelopment without toxicity. Mn2+ release from AuNPs was designed to be pH-responsive so that low pH condition of the cell endosomes can trigger in situ release of Mn2+ from AuNPs after cellular uptake of Mn-incorporated AuNPs (MnAuNPs). Due to the differences in reduction potentials of Mn and Au, only Mn ionized and released while Au remained intact when MnAuNPs were uptaken by cells. Compared to PC12 cells treated with a high concentration of free Mn2+, PC12 cells treated with an equal concentration of MnAuNPs resulted in significantly enhanced cellular neurodevelopment with decreased apoptosis and necrosis. Treatment with a high concentration of free Mn2+ led to an abrupt consumption of a large amount of ATP for the intracellular transport of Mn2+ through the ion channel of the cell membrane and to mitochondrial damage caused by the high intracellular concentration of Mn2+, both of which resulted in cell necrosis and apoptosis. In contrast, MnAuNP-treated cells consumed much smaller amount of ATP for the intracellular transport of MnAuNPs by endocytosis and showed pH-triggered in situ release of Mn2+ from the MnAuNPs in the endosomes of the cells, both of which prevented the cell death caused by ATP depletion and mitochondrial damage. To our knowledge, this is the first report on the use of AuNPs as a vehicle for pH-responsive, intracellular delivery of metal ion, which may open a new window for drug delivery and clinical therapy.

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells.

The BM2 protein of influenza B virus interacts with p53 and inhibits its transcriptional and apoptotic activities.

The influenza virus integral membrane proteins BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for viral replication. M2 ion channel activity is also required to stimulate NLRP3 inflammasome activation by perturbing ionic concentrations in the Golgi. In the present study, we have investigated further the interaction between BM2 and p53 to confirm our previous studies using yeast two-hybrid assays. The interaction between BM2 and p53 was confirmed by GST pull-down, co-immunoprecipitation assays, and confocal microscopy. Expression of BM2 results in down-regulation of p53 mRNA and protein expression in a dose-dependent manner. In addition, we demonstrated that exogenous expression of BM2 functionally blocked p53-mediated transcriptional activity and apoptosis by luciferase reporter assay and TUNEL assay, respectively. Together, the present results indicate that BM2 is able to functionally interact with p53, and provide valuable insights into the modulation of p53 functions by influenza virus.

EGF‑stimulated AKT activation is mediated by EGFR recycling via an early endocytic pathway in a gefitinib‑resistant human lung cancer cell line.

The receptor tyrosine kinase epidermal growth factor receptor (EGFR) and its ligand epidermal growth factor (EGF) are known to play important roles in malignant tumor cells, and the EGFR signaling pathway is one of the most important targets in various tumors, including non-small cell lung cancer (NSCLC). We reported recently that an aberration in certain steps of EGF-stimulated phosphorylated epidermal growth factor receptor (pEGFR) endocytic trafficking from the early endosomes to the late endosomes occurs in the gefitinib-resistant NSCLC cells, in which large amounts of sorting nexin 1 (SNX1) are colocalized with EGFR in the aggregated early endosomes where the internalized pEGFR is also accumulated of these cells. To further investigate the role of SNX1 in EGF‑stimulated pEGFR endocytosis, followed by downstream signaling leading to the activation of phosphatidylinositol 3-kinase (PI3K)--the serine/threonine kinase AKT pathway, we examined the effect of depletion of SNX1 knock-down expression by siRNA and an inhibition of targeting membrane recycling using monensin. Using immunofluorescence, we observed an efficient endocytic transport of pEGFR from early endosomes to late endosomes/lysosomes after EGF-stimulation in the cells transfected with siRNA‑SNX1, whereas the delayed endocytic delivery of pEGFR was evident in the siRNA-control-transfected cells. Furthermore, a large amount of endocytosed pEGFR was accumulated in the presence of monensin in the early endosomes of the SNX1 knock-down cells. In western blot analysis, EGF stimulation of both control and cells transfected with siRNA-SNX1 resulted in rapid phosphorylation of EGFR and enhanced AKT phosphorylation. Monensin-dependent inhibition of AKT phosphorylation was stronger in SNX1 knock-down cells than in controls. In contrast, however, monensin had no effect on AKT phosphorylation triggered by activation of the MET receptor tyrosine kinase. Collectively, we suggest that EGF-stimulated recycling of EGFR to the plasma membrane induces downstream signaling leading to AKT phosphorylation. Suppression of EGFR membrane recycling by SNX1 appears to be critical for the activation of EGFR/PI3K/AKT signaling pathway in human lung cancer cells.