Src Homology 2-containing Inositol Research Articles

SHIP1 deficiency causes inflammation-dependent retardation in skeletal growth.

Inflammation and skeletal homeostasis are closely intertwined. Inflammatory diseases are associated with local and systemic bone loss, and post-menopausal osteoporosis is linked to low-level chronic inflammation. Phosphoinositide-3-kinase signalling is a pivotal pathway modulating immune responses and controlling skeletal health. Mice deficient in Src homology 2-containing inositol phosphatase 1 (SHIP1), a negative regulator of the phosphoinositide-3-kinase pathway, develop systemic inflammation associated with low body weight, reduced bone mass, and changes in bone microarchitecture. To elucidate the specific role of the immune system in skeletal development, a genetic approach was used to characterise the contribution of SHIP1-controlled systemic inflammation to SHIP1-dependent osteoclastogenesis. Lymphocyte deletion entirely rescued the skeletal phenotype in Rag2 -/- /Il2rg -/- /SHIP1 -/- mice. Rag2 -/- /Il2rg -/- /SHIP1 -/- osteoclasts, however, displayed an intermediate transcriptomic signature between control and Rag2 +/+ /Il2rg +/+ /SHIP1 -/- osteoclasts while exhibiting aberrant in vitro development and functions similar to Rag2 +/+ /Il2rg +/+ /SHIP1 -/- osteoclasts. These data establish a cell-intrinsic role for SHIP1 in osteoclasts, with inflammation as the key driver of the skeletal phenotype in SHIP1-deficient mice. Our findings demonstrate the central role of the immune system in steering physiological skeletal development.

PHB2 promotes SHIP2 ubiquitination via the E3 ligase NEDD4 to regulate AKT signaling in gastric cancer

BackgroundProhibitin 2 (PHB2) exhibits opposite functions of promoting or inhibiting tumour across various cancer types. In this study, we aim to investigate its functions and underlying mechanisms in the context of gastric cancer (GC).MethodsPHB2 protein expression levels in GC and normal tissues were examined using western blot and immunohistochemistry. PHB2 expression level associations with patient outcomes were examined through Kaplan–Meier plotter analysis utilizing GEO datasets (GSE14210 and GSE29272). The biological role of PHB2 and its subsequent regulatory mechanisms were elucidated in vitro and in vivo. GC cell viability and proliferation were assessed using MTT cell viability analysis, clonogenic assays, and BrdU incorporation assays, while the growth of GC xenografted tumours was measured via IHC staining of Ki67. The interaction among PHB2 and SHIP2, as well as between SHIP2 and NEDD4, was identified through co-immunoprecipitation, GST pull-down assays, and deletion-mapping experiments. SHIP2 ubiquitination and degradation were assessed using cycloheximide treatment, plasmid transfection and co-immunoprecipitation, followed by western blot analysis.ResultsOur analysis revealed a substantial increase in PHB2 expression in GC tissues compared to adjacent normal tissues. Notably, higher PHB2 levels correlated with poorer patient outcomes, suggesting its clinical relevance. Functionally, silencing PHB2 in GC cells significantly reduced cell proliferation and retarded GC tumour growth, whereas overexpression of PHB2 further enhanced GC cell proliferation. Mechanistically, PHB2 physically interacted with Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) in the cytoplasm of GC cells, thus leading to SHIP2 degradation via its novel E3 ligase NEDD4. It subsequently activated the PI3K/Akt signaling pathway and thus promoted GC cell proliferation.ConclusionsOur findings highlight the importance of PHB2 upregulation in driving GC progression and its association with adverse patient outcomes. Understanding the functional impact of PHB2 on GC growth contributes valuable insights into the molecular underpinnings of GC and may pave the way for the development of targeted therapies to improve patient outcomes.

SHIP1 modulates antimalarial immunity by bridging the crosstalk between type I IFN signaling and autophagy.

Stringent control of the type I interferon (IFN-I) signaling is critical for host immune defense against infectious diseases, yet the molecular mechanisms that regulate this pathway remain elusive. Here, we show that Src homology 2 containing inositol phosphatase 1 (SHIP1) suppresses IFN-I signaling by promoting IRF3 degradation during malaria infection. Genetic ablation of Ship1 in mice leads to high levels of IFN-I and confers resistance to Plasmodium yoelii nigeriensis (P.y.) N67 infection. Mechanistically, SHIP1 promotes the selective autophagic degradation of IRF3 by enhancing K63-linked ubiquitination of IRF3 at lysine 313, which serves as a recognition signal for NDP52-mediated selective autophagic degradation. In addition, SHIP1 is downregulated by IFN-I-induced miR-155-5p upon P.y. N67 infection and severs as a feedback loop of the signaling crosstalk. This study reveals a regulatory mechanism between IFN-I signaling and autophagy, and verifies SHIP1 can be a potential target for therapeutic intervention against malaria and other infectious diseases. IMPORTANCE Malaria remains a serious disease affecting millions of people worldwide. Malaria parasite infection triggers tightly controlled type I interferon (IFN-I) signaling that plays a critical role in host innate immunity; however, the molecular mechanisms underlying the immune responses are still elusive. Here, we discover a host gene [Src homology 2-containing inositol phosphatase 1 (SHIP1)] that can regulate IFN-I signaling by modulating NDP52-mediated selective autophagic degradation of IRF3 and significantly affect parasitemia and resistance of Plasmodium-infected mice. This study identifies SHIP1 as a potential target for immunotherapies in malaria and highlights the crosstalk between IFN-I signaling and autophagy in preventing related infectious diseases. SHIP1 functions as a negative regulator during malaria infection by targeting IRF3 for autophagic degradation.

ACE2 negatively regulates the Warburg effect and suppresses hepatocellular carcinoma progression via reducing ROS-HIF1α activity.

Aerobic glycolysis has pleiotropic roles in the pathogenesis of hepatocellular carcinoma (HCC). Emerging studies revealed key promoters of aerobic glycolysis, however, little is known about its negative regulators in HCC. In this study, an integrative analysis identifies a repertoire of differentially expressed genes (DNASE1L3, SLC22A1, ACE2, CES3, CCL14, GYS2, ADH4, and CFHR3) that are inversely associated with the glycolytic phenotype in HCC. ACE2, a member of the rennin-angiotensin system, is revealed to be downregulated in HCC and predicts a poor prognosis. ACE2 overexpression significantly inhibits the glycolytic flux as evidenced by reduced glucose uptake, lactate release, extracellular acidification rate, and the expression of glycolytic genes. Opposite results are noticed in loss-of-function studies. Mechanistically, ACE2 metabolizes Ang II to Ang-(1-7), which activates Mas receptor and leads to the phosphorylation of Src homology 2-containing inositol phosphatase 2 (SHP-2). SHP2 activation further blocks reactive oxygen species (ROS)-HIF1α signaling. Addition of Ang-(1-7) or the antioxidant N-acetylcysteine compromises in vivo additive tumor growth and aerobic glycolysis induced by ACE2 knockdown. Moreover, growth advantages afforded by ACE2 knockdown are largely glycolysis-dependent. In clinical settings, a close link between ACE2 expression and HIF1α or the phosphorated level of SHP2 is found. Overexpression of ACE2 significantly retards tumor growth in patient-derived xenograft model. Collectively, our findings suggest that ACE2 is a negative glycolytic regulator, and targeting the ACE2/Ang-(1-7)/Mas receptor/ROS/HIF1α axis may be a promising therapeutic strategy for HCC treatment.

IQGAP2 Inhibits Migration and Invasion of Gastric Cancer Cells via Elevating SHIP2 Phosphatase Activity.

Previous studies have shown reduced expression of Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) and its tumor-suppressive role in gastric cancer (GC). However, the precise role of SHIP2 in the migration and invasion of GC cells remains unclear. Here, an IQ motif containing the GTPase-activating protein 2 (IQGAP2) as a SHIP2 binding partner, was screened and identified by co-immunoprecipitation and mass spectrometry studies. While IQGAP2 ubiquitously expressed in GC cells, IQGAP2 and SHIP2 co-localized in the cytoplasm of GC cells, and this physical association was confirmed by the binding of IQGAP2 to PRD and SAM domains of SHIP2. The knockdown of either SHIP2 or IQGAP2 promoted cell migration and invasion by inhibiting SHIP2 phosphatase activity, activating Akt and subsequently increasing epithelial–mesenchymal transition (EMT). Furthermore, knockdown of IQGAP2 in SHIP2-overexpressing GC cells reversed the inhibition of cell migration and invasion by SHIP2 induction, which was associated with the suppression of elevated SHIP2 phosphatase activity. Moreover, the deletion of PRD and SAM domains of SHIP2 abrogated the interaction and restored cell migration and invasion. Collectively, these results indicate that IQGAP2 interacts with SHIP2, leading to the increment of SHIP2 phosphatase activity, and thereby inhibiting the migration and invasion of GC cells via the inactivation of Akt and reduction in EMT.

SHIP-1, a target of miR-155, regulates endothelial cell responses in lung fibrosis.

Src Homology 2-containing Inositol Phosphatase-1 (SHIP-1) is a target of miR-155, a pro-inflammatory factor. Deletion of the SHIP-1 gene in mice caused spontaneous lung inflammation and fibrosis. However, the role and function of endothelial miR-155 and SHIP-1 in lung fibrosis remain unknown. Using whole-body miR-155 knockout mice and endothelial cell-specific conditional miR-155 (VEC-Cre-miR-155 or VEC-miR-155) or SHIP-1 (VEC-SHIP-1) knockout mice, we assessed endothelial-mesenchymal transition (EndoMT) and fibrotic responses in bleomycin (BLM) induced lung fibrosis models. Primary mouse lung endothelial cells (MLEC) and human umbilical vein endothelial cells (HUVEC) with SHIP-1 knockdown were analyzed in TGF-β1 or BLM, respectively, induced fibrotic responses. Fibrosis and EndoMT were significantly reduced in miR-155KO mice and changes in EndoMT markers in MLEC after TGF-β1 stimulation confirmed the in vivo findings. Furthermore, lung fibrosis and EndoMT responses were reduced in VEC-miR-155 mice but significantly enhanced in VEC-SHIP-1 mice after BLM challenge. SHIP-1 knockdown in HUVEC cells resulted in enhanced EndoMT induced by BLM. Meanwhile, these changes involved the PI3K/AKT, JAK/STAT3, and SMAD/STAT signaling pathways. These studies demonstrate that endothelial miR-155 plays an important role in fibrotic responses in the lung through EndoMT. Endothelial SHIP-1 is essential in controlling fibrotic responses and SHIP-1 is a target of miR-155. Endothelial cells are an integral part in lung fibrosis.

Upregulation of SHIP2 participates in the development of breast cancer via promoting Wnt/β-catenin signaling.

PurposeSrc homology 2-containing inositol 5-phosphatase 2 (SHIP2) gene is associated with arthrosclerosis, gastric cancer and diabetes. In this study, we revealed that overexpression of SHIP2 is closely implicated with the development of breast cancer (BC).MethodsThe BC tissue and adjacent cancerous tissue were obtained from BC patients who had underwent mastectomy. BC cells with either overexpression or knockdown of SHIP2 were analyzed to determine cell proliferation, migration, invasion and apoptosis using the CCK-8 assay, colony formation assay, scratch assay, transwell assay and flow cytometry, respectively. A rat BC xenograft model was constructed to explore the role of SHIP2 on tumor growth in vivo.ResultsThe expression levels of SHIP2 in BC tissues and cells were significantly higher than those in adjacent tissues and normal breast cells, respectively. Silencing SHIP2 suppressed BC cells proliferation and promoted apoptosis. Overexpression of SHIP2 enhanced the cells migration/invasion in BC. Moreover, SHIP2 participated in the Wnt/β-catenin pathway by regulating GSK-3β and its downstream genes. β-Catenin activator LiCl could significantly eliminate the effect of si-SHIP2 on BC cells. Moreover, overexpression of SHIP2 increased tumor volume and weight in rat model, and Wnt/β-catenin pathway inhibitor ICG001 reversed the promoting effect of SHIP2 on tumorigenesis.ConclusionUpregulation of SHIP2 could increase the migration, invasion, proliferation, and decrease apoptosis in BC cells. Moreover, SHIP2 participated in the progression of BC via activating the Wnt/β-catenin pathway.

Brain Lipopolysaccharide Preconditioning-Induced Gene Reprogramming Mediates a Tolerance State in Electroconvulsive Shock Model of Epilepsy

There is increasing evidence pointing toward the role of inflammatory processes in epileptic seizures, and reciprocally, prolonged seizures induce more inflammation in the brain. In this regard, effective strategies to control epilepsy resulting from neuroinflammation could be targeted. Based on the available data, preconditioning (PC) with low dose lipopolysaccharide (LPS) through the regulation of the TLR4 signaling pathway provides neuroprotection against subsequent challenge with injury in the brain. To test this, we examined the effects of a single and chronic brain LPS PC, which is expected to lead to reduction of inflammation against epileptic seizures induced by electroconvulsive shock (ECS). A total of 60 male Sprague Dawley rats were randomly assigned to five groups: control, vehicle (single and chronic), and LPS PC (single and chronic). We first recorded the data regarding the behavioral and histological changes. We further investigated the alterations of gene and protein expression of important mediators in relation to TLR4 and inflammatory signaling pathways. Interestingly, significant increased presence of NFκB inhibitors [Src homology 2-containing inositol phosphatase-1 (SHIP1) and Toll interacting protein (TOLLIP)] was observed in LPS-preconditioned animals. This result was also associated with over-expression of IRF3 activity and anti-inflammatory markers, along with down-regulation of pro-inflammatory mediators. Summarizing, the analysis revealed that PC with LPS prior to seizure induction may have a neuroprotective effect possibly by reprogramming the signaling response to injury.

IVIG activates Fc\u03b3RIIB-SHIP1-PIP3 Pathway to stabilize mast cells and suppress inflammation after ICH in mice

Following intracerebral hemorrhage (ICH), the activation of mast cell contributes to brain inflammation and brain injury. The mast cell activation is negatively regulated by an inhibitory IgG-receptor. It’s signals are mediated by SHIP (Src homology 2-containing inositol 5′ phosphatase), in particular SHIP1, which activation leads to hydrolyzation of PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3, leading to the inhibition of calcium mobilization and to the attenuation of mast cell activation. Intravenous immunoglobulin (IVIG) is a FDA-approved drug containing IgG. We hypothesized that IVIG will attenuate the ICH-induced mast cell activation via FcγRIIB/SHIP1 pathway, resulting in a decrease of brain inflammation, protection of the blood-brain-barrier, and improvement of neurological functions after ICH. To prove this hypothesis we employed the ICH collagenase mouse model. We demonstrated that while ICH induced mast cell activation/degranulation, IVIG attenuated post-ICH mast cell activation. Mast cell deactivation resulted in reduced inflammation, consequently attenuating brain edema and improving of neurological functions after ICH. Furthermore using siRNA-induced in vivo knockdown approach we demonstrated that beneficial effects of IVIG were mediated, at least partly, via SHIP1/PIP3 pathway. We conclude that IVIG treatment represents a promising therapeutic approach potentially able to decrease mortality and morbidity after ICH in experimental models.

Circulating SHIP2 mRNA as a novel biomarker in the diagnosis and prognosis of gastric cancer.

Previous studies showed the aberrant expression of Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) in GC tissue. However, the exact role of circulating SHIP2 in GC remains unclear. The aim of this manuscript was to analyze potential diagnostic and prognostic value of circulating SHIP2 levels in GC. Circulating SHIP2 expression was detected in the plasma of 156 GC patients and 60 healthy controls by qRT-PCR. The receiver operating characteristic (ROC) curves were plotted to explore the reliability of circulating SHIP2 in detecting GC. Survival curves were estimated using the Kaplan-Meier method, and differences between them were evaluated by the log-rank test. The influence of each variable on survival was examined by the Cox multivariate regression analysis. Our research showed that the expression levels of circulating SHIP2 in plasma of GC patients were lower than in healthy controls (p < 0.05). Decreased circulating SHIP2 mRNA expression was negatively correlated with clinical stage (p = 0.004), lymph node metastasis (p= 0.003) and distant metastasis (p = 0.025). ROC curve analysis showed that circulating SHIP2 may be a useful marker for discriminating cases from healthy controls. In addition, patients with low circulating SHIP2 mRNA level had poorer overall survival than those with high circulating SHIP2 mRNA level (p = 0.006). Moreover, multivariate analysis indicated that the level of circulating SHIP2 mRNA expression was an independent prognostic indicator (p = 0.005) for the survival of patients with GC. The present study indicated that decreased plasma SHIP2 mRNA level might be a novel diagnostic and prognostic biomarker for GC patients. This conclusion should be further assessed in randomized clinical trials.

Role of lipid inositol phosphatase SHIP-1 in Germinal Center B cell selection and function

Abstract Germinal Centers (GC) are sites within secondary lymphoid organs in which B cells undergo proliferation and differentiation. The PI3K signaling pathway is a central modulator of GC B cells (GCBC) selection and function. The PI3K pathway can be regulated by several enzymes including Src homology 2-containing inositol 5’-phosphatase (SHIP-1). SHIP-1 is known to be hyper-phosphorylated in GCBCs. Upon activation, SHIP-1 hydrolyzes phosphatidyl inositol triphosphate (PIP3) to generate phosphatidyl inositol 3,4 biphosphate (PIP2). PIP2 can bind to signaling proteins that are crucial to BCR signaling such as TAPP1/2 and Akt/PKB. To ascertain the role of SHIP-1 in survival and selection of GCBC we used a tamoxifen inducible Cre system to delete SHIP-1 from B cells during an ongoing GC response to NP-CGG. SHIP-1 deletion led to bi-modal expression of SHIP-1 in GCBC expressing intermediate (SHIP-1 int) and low levels of SHIP-1 (SHIP-1 lo) compared to WT controls (SHIP-1 hi). SHIP-1 int GCBC showed reduced phosphorylation of signaling proteins S6 and increased phosphorylation of Btk upon BCR crosslinking with anti-u antibody. SHIP-1 lo cells were un-responsive to BCR crosslinking and had the highest frequency of dead cells and Caspase 3 positive cells among all 3 groups. This data suggests that SHIP-1 expression and activity is crucial to GCBC signaling and survival. The role of PIP2 in regulating BCR signaling in GCBC remains to be elucidated.

Decreased Sp1 Expression Mediates Downregulation of SHIP2 in Gastric Cancer Cells.

Past studies have shown that the Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) is commonly downregulated in gastric cancer, which contributes to elevated activation of PI3K/Akt signaling, proliferation and tumorigenesis of gastric cancer cells. However, the mechanisms underlying the reduced expression of SHIP2 in gastric cancer remain unclear. While gene copy number variation analysis and exon sequencing indicated the absence of genomic alterations of SHIP2, bisulfite genomic sequencing (BGS) showed promoter hypomethylation of SHIP2 in gastric cancer cells. Analysis of transcriptional activity of SHIP2 promoter revealed Specificity protein 1 (Sp1) was responsible for the regulation of SHIP2 expression in gastric cancer cells. Furthermore, Sp1 expression, but not Sp3, was frequently downregulated in gastric cancer compared with normal gastric mucosa, which was associated with a paralleled reduction in SHIP2 levels in gastric cancer. Moreover, overexpression of Sp1 inhibited cell proliferation, induced apoptosis, suppressed cell motility and invasion in gastric cancer cells in vitro, which was, at least in part, due to transcriptional activation of SHIP2 mediated by Sp1, thereby inactivating Akt. Collectively, these results indicate that decreased expression of transcription factor Sp1 contributes to suppression of SHIP2 in gastric cancer cells.

The early activation of memory B cells from Wiskott-Aldrich syndrome patients is suppressed by CD19 downregulation

AbstractWiskott-Aldrich syndrome (WAS) pediatric patients exhibit a deficiency in humoral immune memory. However, the mechanism by which Wiskott-Aldrich syndrome protein (WASP) regulates the differentiation and activation of memory B cells remains elusive. Here we examine the early activation events of memory B cells from the peripheral blood mononuclear cells of WAS patients and age-matched healthy controls (HCs) using total internal reflection fluorescence microscopy. In response to stimulation through the B-cell receptor (BCR), memory B cells from HCs showed significantly higher magnitudes of BCR clustering and cell spreading than naive B cells from the same individuals. This was associated with increases in CD19 recruitment to the BCR and the activation of its downstream signaling molecule Btk and decreases in FcγRIIB recruitment and the activation of its downstream molecule Src homology 2-containing inositol 5′ phosphatase (SHIP). However, these enhanced signaling activities mediated by CD19 and Btk are blocked in memory B cells from WAS patients, whereas the activation of FcγRIIB and SHIP was increased. Although the expression levels of CD19, Btk, and FcγRIIB did not change between CD27− and CD27+ B cells of HCs, the protein and mRNA levels of CD19 but not Btk and FcγRIIB were significantly reduced in both CD27− and CD27+ B cells of WAS patients, compared with those of HCs. Overall, our study suggests that WASP is required for memory B-cell activation, promoting the activation by positive regulating CD19 transcription and CD19 recruitment to the BCR.

Targeting miR-155 to Treat Experimental Scleroderma.

Scleroderma is a refractory autoimmune skin fibrotic disorder. Alterations of microRNAs in lesional skin could be a new approach to treating the disease. Here, we found that expression of miR-155 was up regulated in lesional skin tissue from patients with either systemic or localized scleroderma, and correlated with fibrosis area. Then we demonstrated the potential of miR-155 as a therapeutic target in pre-clinical scleroderma models. MiR-155−/− mice were resistant to bleomycin induced skin fibrosis. Moreover, topical antagomiR-155 could effectively treat mice primed with subcutaneous bleomycin. In primary skin fibroblast, miR-155 silencing could inhibit collagen synthesis function, as well as signaling intensity of two pro-fibrotic pathways, Wnt/β-catenin and Akt, simultaneously. We further showed that miR-155 could regulate the two pathways via directly targeting casein kinase 1α (CK1α) and Src homology 2-containing inositol phosphatase-1 (SHIP-1), as previous reports. Mice with miR-155 knockout or topical antagomir-155 treatment showed inhibited Wnt/β-catenin and Akt signaling in skin upon bleomycin challenge. Together, our data suggest the potential of miR-155 silencing as a promising treatment for dermal fibrosis, especially in topical applications.

Suppression of SHIP2 contributes to tumorigenesis and proliferation of gastric cancer cells via activation of Akt.

The Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) is implicated in diabetes, arthrosclerosis, and cancer. However, the role of SHIP2 in human gastric cancer remains unclear. The expression levels of SHIP2 in gastric cancer tissues, a panel of gastric cancer cell lines, and normal gastric epithelial cells were analyzed by immunohistochemistry (IHC), Western blot, and real-time quantitative RT-PCR (qRT-PCR). Gastric cancer cells with either overexpressed SHIP2 or co-overexpressed SHIP2 and Akt were analyzed to determine cell proliferation, colony formation, apoptosis, cell migration, and invasion assays. Normal gastric epithelial cells with knockdown SHIP2 or co-knockdown SHIP2 and Akt were subjected by anchorage-independent growth assays. The effect of SHIP2 on tumor growth in vivo was detected by xenograft tumorigenesis assays. SHIP2 was commonly downregulated in gastric cancer compared with normal gastric mucosa, and overexpression of SHIP2 inhibited cell proliferation, induced apoptosis, suppressed cell motility and invasion in gastric cancer cells in vitro, and retarded the growth of xenograft gastric tumors in vivo, while knockdown of SHIP2 in normal gastric epithelial cells promoted anchorage-independent growth. Moreover, overexpression of SHIP2 inactivated Akt, and upregulated p21, p27, and the pro-apoptotic protein Bim. Restoring Akt activation in gastric cancer cells largely blocked the inhibition of PI3K/Akt signaling by SHIP2 and reversed the inhibitory effect of SHIP2 on tumorigenesis and proliferation. This study demonstrates, for the first time, that SHIP2 is frequently downregulated in gastric cancer, and reduced SHIP2 expression promotes tumorigenesis and proliferation of gastric cancer via activation of the PI3K/Akt signaling.

Micro-RNA-155 deficiency prevents alcohol-induced serum endotoxin increase and small bowel inflammation in mice.

Chronic alcohol impairs gut barrier function and induces inflammatory cytokines. The effects of acute alcohol binge on the gut are partially understood. Micro-RNA-155 (miR-155), a modulator of cytokine and T-cell immune response in the gut, stabilizes tumor necrosis factor-α (TNFα) mRNA. Here, we investigated the role of the inflammation modulator miR-155 as well as the effects of acute binge and chronic alcohol feeding in the small bowel (SB) in mice. For the acute alcohol binge, wild-type (WT) mice received 5g/kg 50% alcohol/d or equal amount of water oral gavage for 3days. WT and miR-155-deficient (miR-155-knockout [KO]) mice received ethanol containing Lieber-DeCarli or isocaloric control diet for 5weeks. MiR-155, antimicrobial peptide, regenerating islet-derived 3-beta (Reg3b), inflammation markers, Src homology 2-containing inositol phosphatase-1 (SHIP1), TNFα, and nuclear factor-κB (NF-κB) were measured in proximal intestinal tissue. Endotoxin was measured in the serum. Acute alcohol binge enhanced, whereas chronic alcohol feeding decreased, Reg3b mRNA and protein levels in the SB. Both acute binge and chronic alcohol feeding increased serum endotoxin levels, intestinal NF-κB activation and TNFα mRNA levels. However, TNFα protein and miR-155 were increased only after chronic alcohol feeding in the SB. Furthermore, miR-155-KO mice were protected from chronic alcohol-induced increase in serum endotoxin, intestinal TNFα, and NF-κB activation. Also, alcohol-fed miR-155-KO mice had no decrease of Reg3b and SHIP1 levels. These results demonstrate that both acute binge and chronic ethanol administration result in increased serum-endotoxin levels. Our study identifies a novel role for miR-155 in chronic alcohol-induced intestinal inflammation and barrier dysfunction.

MicroRNA 155 Regulates Japanese Encephalitis Virus-Induced Inflammatory Response by Targeting Src Homology 2-Containing Inositol Phosphatase 1

MicroRNAs (miRNAs) are single-stranded small RNA molecules that regulate various cellular processes. miRNA 155 (miR-155) regulates various aspects of innate and adaptive immune responses and plays a key role in various viral infections and the resulting neuroinflammation. The present study evaluated the involvement of miR-155 in modulating Japanese encephalitis virus (JEV)-induced neuroinflammation. We observed that miR-155 expression was upregulated during JEV infection of mouse primary microglia, the BV-2 microglia cell line, and in both mouse and human brains. In vitro and in vivo knockdown of miR-155 minimized JEV-induced inflammatory responses. In the present study, we confirmed targeting of the Src homology 2-containing inositol phosphatase 1 (SHIP1) 3' untranslated region (UTR) by miR-155 in the context of JEV infection. Inhibition of SHIP1 by miR-155 resulted in higher beta interferon (IFN-β) and proinflammatory cytokine production through activation of TANK-binding kinase 1 (TBK-1). Based on these observations, we conclude that miR-155 modulates the neuroinflammatory response during JEV infection via negative regulation of SHIP1 expression. Thus, modulation of miR-155 could be a novel strategy to regulate JEV-induced neuroinflammation. Japanese encephalitis virus (JEV), a member of the family Flaviviridae that causes Japanese encephalitis (JE), is the most common mosquito-borne encephalitis virus in the Asia-Pacific region. The disease is feared, as currently there are no specific antiviral drugs available. JEV targets the central nervous system, leading to high mortality and neurological and psychiatric sequelae in some of those who survive. The level of inflammation correlates well with the clinical outcome in patients. Recently, microRNA (miRNA), a single-stranded noncoding RNA, has been implicated in various brain disorders. The present study investigates the role of miRNA in JEV-induced neuroinflammation. Our results show that miRNA 155 (miR-155) targets the Src homology 2-containing inositol phosphatase 1 (SHIP1) protein and promotes inflammation by regulating the NF-κB pathway, increasing the expression of various proinflammatory cytokines and the antiviral response. Thus, miR-155 is a potential therapeutic target to develop antivirals in JE and other brain disorders where inflammation plays a significant role in disease progression.

Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5′-phosphatase interaction with c-Abl

It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. Human EndoC-βH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-βH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-βH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total β-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

Potential role of histamine releasing factor (HRF) as a therapeutic target for treating asthma and allergy

Histamine releasing factor (HRF), also known as translationally controlled tumor protein (TCTP), is a highly conserved, ubiquitous protein that has both intracellular and extracellular functions. Here, we will highlight the history of the molecule, its clinical implications with a focus on its extracellular functioning, and its potential role as a therapeutic target in asthma and allergy. The cells and cytokines produced when stimulated or primed by HRF/TCTP are detailed as well as the downstream signaling pathway that HRF/TCTP elicits. While it was originally thought that HRF/TCTP interacted with IgE, the finding that cells not binding IgE also respond to HRF/TCTP called this interaction into question. HRF/TCTP, or at least its mouse counterpart, appears to interact with some, but not all IgE and IgG molecules. HRF/TCTP has been shown to activate multiple human cells including basophils, eosinophils, T cells, and B cells. Since many of the cells activated by HRF/TCTP participate in the allergic response, extracellular functions of HRF/TCTP may exacerbate the allergic, inflammatory cascade. Particularly exciting is that small molecule agonists of Src homology 2-containing inositol phosphatase-1 have been shown to modulate the phosphoinositide 3-kinase/AKT pathway and may control inflammatory disorders. This review discusses this possibility in light of HRF/TCTP.

A Phosphatidylinositol Lipids System, Lamellipodin, and Ena/VASP Regulate Dynamic Morphology of Multipolar Migrating Cells in the Developing Cerebral Cortex

In the developing mammalian cerebral cortex, excitatory neurons are generated in the ventricular zone (VZ) and subventricular zone; these neurons migrate toward the pial surface. The neurons generated in the VZ assume a multipolar morphology and remain in a narrow region called the multipolar cell accumulation zone (MAZ) for ∼24 h, in which they extend and retract multiple processes dynamically. They eventually extend an axon tangentially and begin radial migration using a migratory mode called locomotion. Despite the potential biological importance of the process movement of multipolar cells, the molecular mechanisms remain to be elucidated. Here, we observed that the processes of mouse multipolar cells were actin rich and morphologically resembled the filopodia and lamellipodia in growth cones; thus, we focused on the actin-remodeling proteins Lamellipodin (Lpd) and Ena/vasodilator-stimulated phosphoprotein (VASP). Lpd binds to phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P₂] and recruits Ena/VASP, which promotes the assembly of actin filaments, to the plasma membranes. In situ hybridization and immunohistochemistry revealed that Lpd is expressed in multipolar cells in the MAZ. The functional silencing of either Lpd or Ena/VASP decreased the number of primary processes. Immunostaining and a Förster resonance energy transfer analysis revealed the subcellular localization of PI(3,4)P₂ at the tips of the processes. A knockdown experiment and treatment with an inhibitor for Src homology 2-containing inositol phosphatase-2, a 5-phosphatase that produces PI(3,4)P₂ from phosphatidylinositol (3,4,5)-triphosphate, decreased the number of primary processes. Our observations suggest that PI(3,4)P₂, Lpd, and Ena/VASP are involved in the process movement of multipolar migrating cells.