SH2 Domain-containing Inositol 5-phosphatase Research Articles

Discovery of aurones bearing two amine functionalities as SHIP2 inhibitors with insulin-sensitizing effect in rat myotubes.

Pharmacological inhibition of the SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) by small-molecule compounds presents an attractive approach to modulate insulin sensitivity. Few drug-like SHIP2 inhibitors have been discovered to date. A series of aurones incorporating key motifs from known SHIP2 inhibitors were synthesized and evaluated for SHIP2-inhibiting activity against a recombinant SHIP2 protein in vitro. Three aurones that inhibited SHIP2 at 15-50 μM were identified. These aurone inhibitors required two amine functionalities, one at ring A and a second at ring B for good inhibitory activity as exemplified by 12a. Mechanistically, molecular dynamics simulations revealed 12a to preferably bind to an allosteric site, restricting the motion of the flexible L4 loop required for SHIP2 phosphatase activity. Additionally, a basic piperidine moiety of 12a interacted with an aspartate residue proximal to the site. At 20-40 μM, 12a significantly enhanced glucose uptake in rat myotubes via increased Akt phosphorylation. 12a showed good permeability across the Caco-2 cell monolayer supporting the aurone chemotype as a new lead to develop drug-like, oral insulin sensitizers.

Regulation of microglial responses after pediatric traumatic brain injury: exploring the role of SHIP-1.

Severe traumatic brain injury (TBI) is the world's leading cause of permanent neurological disability in children. TBI-induced neurological deficits may be driven by neuroinflammation post-injury. Abnormal activity of SH2 domain-containing inositol 5' phosphatase-1 (SHIP-1) has been associated with dysregulated immunological responses, but the role of SHIP-1 in the brain remains unclear. The current study investigated the immunoregulatory role of SHIP-1 in a mouse model of moderate-severe pediatric TBI. SHIP-1+/- and SHIP-1-/- mice underwent experimental TBI or sham surgery at post-natal day 21. Brain gene expression was examined across a time course, and immunofluorescence staining was evaluated to determine cellular immune responses, alongside peripheral serum cytokine levels by immunoassays. Brain tissue volume loss was measured using volumetric analysis, and behavior changes both acutely and chronically post-injury. Acutely, inflammatory gene expression was elevated in the injured cortex alongside increased IBA-1 expression and altered microglial morphology; but to a similar extent in SHIP-1-/- mice and littermate SHIP-1+/- control mice. Similarly, the infiltration and activation of CD68-positive macrophages, and reactivity of GFAP-positive astrocytes, was increased after TBI but comparable between genotypes. TBI increased anxiety-like behavior acutely, whereas SHIP-1 deficiency alone reduced general locomotor activity. Chronically, at 12-weeks post-TBI, SHIP-1-/- mice exhibited reduced body weight and increased circulating cytokines. Pro-inflammatory gene expression in the injured hippocampus was also elevated in SHIP-1-/- mice; however, GFAP immunoreactivity at the injury site in TBI mice was lower. TBI induced a comparable loss of cortical and hippocampal tissue in both genotypes, while SHIP-1-/- mice showed reduced general activity and impaired working memory, independent of TBI. Together, evidence does not support SHIP-1 as an essential regulator of brain microglial morphology, brain immune responses, or the extent of tissue damage after moderate-severe pediatric TBI in mice. However, our data suggest that reduced SHIP-1 activity induces a greater inflammatory response in the hippocampus chronically post-TBI, warranting further investigation.

Regulation of Microglial Signaling by Lyn and SHIP-1 in the Steady-State Adult Mouse Brain.

Chronic neuroinflammation and glial activation are associated with the development of many neurodegenerative diseases and neuropsychological disorders. Recent evidence suggests that the protein tyrosine kinase Lyn and the lipid phosphatase SH2 domain-containing inositol 5' phosphatase-1 (SHIP-1) regulate neuroimmunological responses, but their homeostatic roles remain unclear. The current study investigated the roles of Lyn and SHIP-1 in microglial responses in the steady-state adult mouse brain. Young adult Lyn-/- and SHIP-1-/- mice underwent a series of neurobehavior tests and postmortem brain analyses. The microglial phenotype and activation state were examined by immunofluorescence and flow cytometry, and neuroimmune responses were assessed using gene expression analysis. Lyn-/- mice had an unaltered behavioral phenotype, neuroimmune response, and microglial phenotype, while SHIP-1-/- mice demonstrated reduced explorative activity and exhibited microglia with elevated activation markers but reduced granularity. In addition, expression of several neuroinflammatory genes was increased in SHIP-1-/- mice. In response to LPS stimulation ex vivo, the microglia from both Lyn-/- and SHIP-1-/- showed evidence of hyper-activity with augmented TNF-α production. Together, these findings demonstrate that both Lyn and SHIP-1 have the propensity to control microglial responses, but only SHIP-1 regulates neuroinflammation and microglial activation in the steady-state adult brain, while Lyn activity appears dispensable for maintaining brain homeostasis.

Mechanisms controlling membrane recruitment and activation of the autoinhibited SHIP1 inositol 5-phosphatase

Signal transduction downstream of growth factor and immune receptor activation relies on the production of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3) lipids by PI3K. Regulating the strength and duration of PI3K signaling in immune cells, Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) controls the dephosphorylation of PI(3,4,5)P3 to generate phosphatidylinositol-(3,4)-bisphosphate. Although SHIP1 has been shown to regulate neutrophil chemotaxis, B-cell signaling, and cortical oscillations in mast cells, the role that lipid and protein interactions serve in controlling SHIP1 membrane recruitment and activity remains unclear. Using single-molecule total internal reflection fluorescence microscopy, we directly visualized membrane recruitment and activation of SHIP1 on supported lipid bilayers and the cellular plasma membrane. We find that localization of the central catalytic domain of SHIP1 is insensitive to dynamic changes in PI(3,4,5)P3 and phosphatidylinositol-(3,4)-bisphosphate both invitro and invivo. Very transient SHIP1 membrane interactions were detected only when membranes contained a combination of phosphatidylserine and PI(3,4,5)P3 lipids. Molecular dissection reveals that SHIP1 is autoinhibited with the N-terminal Src homology 2 domain playing a critical role in suppressing phosphatase activity. Robust SHIP1 membrane localization and relief of autoinhibition can be achieved through interactions with immunoreceptor-derived phosphopeptides presented either in solution or conjugated to a membrane. Overall, this work provides new mechanistic details concerning the dynamic interplay between lipid-binding specificity, protein-protein interactions, and the activation of autoinhibited SHIP1.

SHIP1 Controls Internal Platelet Contraction and αIIbβ3 Integrin Dynamics in Early Platelet Activation.

The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is known to dephosphorylate PtdIns(3,4,5)P3 into PtdIns(3,4)P2 and to interact with several signaling proteins though its docking functions. It has been shown to negatively regulate platelet adhesion and spreading on a fibrinogen surface and to positively regulate thrombus growth. In the present study, we have investigated its role during the early phase of platelet activation. Using confocal-based morphometric analysis, we found that SHIP1 is involved in the regulation of cytoskeletal organization and internal contractile activity in thrombin-activated platelets. The absence of SHIP1 has no significant impact on thrombin-induced Akt or Erk1/2 activation, but it selectively affects the RhoA/Rho-kinase pathway and myosin IIA relocalization to the cytoskeleton. SHIP1 interacts with the spectrin-based membrane skeleton, and its absence induces a loss of sustained association of integrins to this network together with a decrease in αIIbβ3 integrin clustering following thrombin stimulation. This αIIbβ3 integrin dynamics requires the contractile cytoskeleton under the control of SHIP1. RhoA activation, internal platelet contraction, and membrane skeleton integrin association were insensitive to the inhibition of PtdIns(3,4,5)P3 synthesis or SHIP1 phosphatase activity, indicating a role for the docking properties of SHIP1 in these processes. Altogether, our data reveal a lipid-independent function for SHIP1 in the regulation of the contractile cytoskeleton and integrin dynamics in platelets.

JAK2-V617F is a negative regulation factor of SHIP1 protein and thus influences the AKT signaling pathway in patients with Myeloproliferative neoplasm (MPN).

Myeloproliferative neoplasms (MPN) are a group of chronic haematological disorders. At the molecular level of MPN cells, the gain-of-function mutation V617F of the Janus kinase 2 (JAK2) leads to a constitutive activation of the downstream signaling cascade and is a conventional criteria for diagnosis. Here, the functional role of the tumor suppressor SHIP1 (SH2 domain containing inositol-5 phosphatase 1) in the pathogenesis of MPNs was investigated. Primary blood samples of MPN-patients were analysed using Western Blot technique regarding the level of SHIP1 expression. Moreover, SHIP1 and SHIP1-mutations were lentivirally transduced in the JAK2-V617F-positive UKE-1 cell line and expression was monitored over time. In addition, we examined SHIP1 reconstitution by inhibition of JAK2-V617F. Furthermore, we transfected SHIP1-expressing cells with a JAK2-V617F respectively a BCR-ABL construct and investigated changes in SHIP1 expression. Four out of five MPN-patient samples showed a loss or a reduction in SHIP1 expression. We identified JAK2 as a negative regulator of SHIP1 expression in MPN cells and inhibition of JAK2-V617F implicates a reconstituted SHIP1 expression. This is significant because SHIP1 negatively regulates the AKT signaling pathway and in consequence the reconstitution of SHIP1 expression leads to a decreased cell growth. Moreover, we examined the impact of SHIP1 and patient-derived SHIP1-mutations on AKT phosphorylation and show the benefit of a combined therapy in MPN cells with inhibitors of the AKT/mTOR pathway. In summary, the data suggest that SHIP1 may play a role during the development of MPNs and could be the basis for establishing a targeted therapy.

Strategy for Leukemia Treatment Targeting SHP-1,2 and SHIP.

Protein tyrosine phosphatases (PTPs) are modulators of cellular functions such as differentiation, metabolism, migration, and survival. PTPs antagonize tyrosine kinases by removing phosphate moieties from molecular signaling residues, thus inhibiting signal transduction. Two PTPs, SHP-1 and SHP-2 (SH2 domain-containing phosphatases 1 and 2, respectively) and another inhibitory phosphatase, SH2 domain-containing inositol phosphatase (SHIP), are essential for cell function, which is reflected in the defective phenotype of mutant mice. Interestingly, SHP-1, SHP-2, and SHIP mutations are identified in many cases of human leukemia. However, the impact of these phosphatases and their mutations regarding the onset and progression of leukemia is controversial. The ambiguity of the role of these phosphatases imposes challenges on the development of targeting therapies for leukemia. This fundamental problem, confronted by the expanding investigational field of leukemia, will be addressed in this review, which will include a discussion of the molecular mechanisms of SHP-1, SHP-2, and SHIP in normal hematopoiesis and their role in leukemia. Clinical development of leukemic therapies achieved by targeting these phosphatases will be addressed as well.

An auxiliary binding interface of SHIP2-SH2 for Y292-phosphorylated FcγRIIB reveals diverse recognition mechanisms for tyrosine-phosphorylated receptors involved in different cell signaling pathways.

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) plays an essential role in regulating phosphatidylinositol level in human cell, and is recruited to many phosphotyrosine (pY)-dependent signal transduction pathways by the SH2 domain. In immunity signaling, immunoreceptor FcγRIIB binds to SHIP2-SH2 via its Y292-phosphorylated immunoreceptor tyrosine-based inhibitory motif (ITIM) and transmits inhibitory signal, which regulates B cell and neuronal cell activity and is associated with immune diseases and Alzheimer's disease. To date, the interaction between SHIP2 and FcγRIIB has not been analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Y292-phosphorylated FcγRIIB-ITIM was analyzed using NMR spectroscopy. The results demonstrated that SHIP2-SH2 mainly utilizes two regions including a pY-binding pocket and a specificity pocket formed by βD, βE, and EF-loop, to bind with FcγRIIB-ITIM in high affinity. In addition to the two regions, the BG-loop of SHIP2-SH2 functions as an auxiliary interface enhancing affinity. By comparing the binding of SHIP2-SH2 with ligands from FcγRIIB and c-MET, a hepatocyte growth factor receptor associated with tumorigenesis, significant differences in interface and affinity were found, suggesting that SHIP2-SH2 applies diverse patterns for binding to different ligand proteins. Moreover, S49, S51, and R70 of SHIP2 were identified to mediate the binding of both FcγRIIB and c-MET, while R28 and Q107 were found to only participate in the binding of c-MET and FcγRIIB respectively. Taken together, this study reveals the diverse mechanisms of SHIP2-SH2 for recognizing different ligands, and provides important clues for selectively manipulating various signaling pathways and specific drug design.

A6 THE ROLE OF TUFT CELLS IN INTESTINAL HOMEOSTASIS

Abstract Background Intestinal epithelial cells may actively regulate homeostasis by recognizing and responding to extracellular signals. One of these cell types, tuft cells, has been proposed to have a role in secretion, absorption, and reception. However, their role in the intestine has not been fully characterized. We have found that tuft cells express the SH2 domain-containing inositol 5’-phosphatase (SHIP), which was formerly thought to be restricted to hematopoietic cells. SHIP negatively regulates PI3K-mediated cell growth, proliferation, and activation. Tuft cells secrete IL-25, which activates group 2 innate lymphoid cells (ILC2s), leading to type 2 immune responses. Tuft cells may contribute to inflammation in the intestine by increasing ILC2 numbers and/or activation, leading to type II inflammation. Aims My hypothesis is that SHIP inhibits tuft cell responses to innate immune stimuli by limiting PI3K activation. Moreover, SHIP deficiency will increase tuft cell responses to commensal microbes, causing ILC2-mediated type II inflammation. To investigate the role of SHIP in tuft cell responses in vivo, I will use a tuft cell-specific SHIP deficient mouse in the dextran sodium sulfate (DSS)-induced colitis model. Methods We created a mouse deficient in SHIP only in intestinal tuft cells (Fabpcre x SHIPfl/fl) to investigate the impact of SHIP deficiency in tuft cells on responses to luminal microbes. Tuft cell-specific SHIP deficient mice (8-week-old) and their wild type littermates were subjected to DSS-induced colitis for 7 days. Clinical disease activity was monitored daily and gross pathology, including total colon length, was examined at the experimental endpoint. The concentrations of pro-inflammatory type I and type II cytokines were assessed in colonic tissue homogenates via ELISA. Results During DSS-induced colitis, mice with SHIP deficient tuft cells had increased disease activity compared to their wild type littermates, particularly evident in their weight loss. Mice with SHIP deficient tuft cells also had significantly shorter colons than their wild type littermates. IL-25 concentrations (produced by tuft cells) were increased in full thickness colon homogenates from mice with SHIP deficient tuft cells. In contrast, pro-inflammatory cytokines IL-1β, IL-6, and TNF did not differ between genotypes. Thus, increased tuft cell activity due to SHIP deficiency correlated with increased disease severity during DSS-induced colitis. Conclusions SHIP deficiency in intestinal tuft cells leads to increased tuft cell activity and exacerbated colitis during DSS treatment. Tuft cells may contribute to inflammation via IL-25 production, leading to increased type II inflammation by ILC2s. In future studies, we will target IL-25 in this model to determine whether increased tuft cell IL-25 production plays a causal role in disease exacerbation. Funding Agencies NSERC

Resveratrol suppresses the reprogramming of macrophages into an endotoxin-tolerant state through the activation of AMP-activated protein kinase

Resveratrol has been reported to have beneficial effects on sepsis by regulating the inflammatory response. However, it remains unclear if resveratrol plays a role in the development of endotoxin tolerance. Treatment with resveratrol in macrophages stimulated with primary lipopolysaccharide (LPS) resulted in the increased production of TNF-α and IL-6 induced by a 2nd dose of LPS (by 74.5 ± 12.9% and 63.4 ± 12%, respectively, compared to untreated cells, P < 0.05). This effect was inhibited by compound C, an AMPK inhibitor, and STO609, a calcium/calmodulin-dependent protein kinase-kinase (CaMKK) inhibitor. Resveratrol diminished the expression of interleukin-1 receptor-associated kinase M (IRAK-M) and Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) by prolonging the exposure of cells to LPS (by 60.8 ± 16.3% and 70.3 ± 18.1%, respectively, compared to LPS only). The effect of resveratrol on the LPS-induced expression of IRAK-M and SHIP1 was inhibited by compound C or STO609. After a 2nd dose of LPS, resveratrol increased phosphorylation of ERK1/2, p38, and JNK in endotoxin tolerant macrophages. In vivo systemic administration of resveratrol prevented a significant increase in mortality rate by cecal ligation and puncture in LPS-induced endotoxin-tolerant mice. These results indicate that resveratrol induces AMPK activation through the Ca2+/CaMKKβ pathway and suppresses the development of endotoxin tolerance by inhibiting LPS-induced expression of IRAK-M and SHIP1.

Inhibitory Receptor Trap: A Platform for Discovery of Inhibitory Receptors That Utilize Inositol Lipid and Phosphotyrosine Phosphatase Effectors.

Among the areas of most impactful recent progress in immunology is the discovery of inhibitory receptors and the subsequent translation of this knowledge to the clinic. Although the original and canonical member of this family is FcγRIIB, more recent studies defined PD1 as an inhibitory receptor that constrains T cell immunity to tumors. These studies led to development of “checkpoint blockade” immunotherapies (CBT) for cancers in which PD1 interactions with its ligand are blocked. Unfortunately, although very effective in some patients, only a small proportion respond to this therapy. This suggests that additional as yet undescribed inhibitory receptors exist, which could be exploited. Here, we describe a new platform, termed inhibitory receptor trap (IRT), for discovery of members of this family. The approach takes advantage of the fact that many of the known inhibitory receptors mediate signaling by phospho-immunoreceptor tyrosine-based inhibition motif (ITIM) mediated recruitment of Src Homology 2 (SH2) domain-containing phosphatases including the SH2 domain-containing inositol phosphatase SHIP1 encoded by the INPP5D gene and the SH2 domain-containing phosphotyrosine phosphatases SHP1 and SHP2 encoded by the PTPN6 and PTPN11 genes respectively. Here, we describe the IRT discovery platform in which the SH2 domains of inhibitory phosphatases are used for affinity-based isolation and subsequent identification of candidate effectors via immunoblotting and high sensitivity liquid chromatography–mass spectrometry. These receptors may represent alternative targets that can be exploited for improved CBT. Salient observations from these studies include the following: SH2 domains derived from the respective phosphatases bind distinct sets of candidates from different cell types. Thus, cells of different identity and different activation states express partially distinct repertoires of up and downstream phosphatase effectors. Phosphorylated PD1 binds not only SHP2 but also SHIP1, thus the latter may be important in its inhibitory function. B cell antigen receptor signaling leads predominantly to CD79 mono-phosphorylation as indicated by much greater binding to LynSH2 than Syk(SH2)2. This balance of ITAM mono- versus bi-phosphorylation likely tunes signaling by varying activation of inhibitory (Lyn) and stimulatory (Syk) pathways.

Streptococcus pneumoniae Endopeptidase O Promotes the Clearance of Staphylococcus aureus and Streptococcus pneumoniae via SH2 Domain-Containing Inositol Phosphatase 1-Mediated Complement Receptor 3 Upregulation.

Increasing evidences demonstrate that microorganism and their products protect against bacterial and viral pathogens through various mechanisms including immunomodulation. Streptococcus pneumoniae endopeptidase O (PepO), a pneumococcal virulence protein, has been proven to enhance the phagocytosis of Staphylococcus aureus and Streptococcus pneumoniae by macrophages in our previous study, where we detected the down regulation of SH2 domain-containing inositol phosphatase 1 (SHIP1) and the up regulation of complement receptor 3 (CR3) in PepO-stimulated macrophages. In the present study, using SHIP1 over-expression plasmid and CR3 siRNA, we proved that the down regulation of SHIP1 and the up regulation of CR3 mediate the enhanced phagocytosis of S. aureus and S. pneumoniae by PepO-stimulated macrophages. The down regulation of SHIP1 also mediates the up regulation of CR3. To further determine whether PepO protects against respiratory pathogens, we constructed a mouse model with intranasal infection of S. aureus or S. pneumoniae and found that PepO significantly promoted their clearance. The down regulation of SHIP1 and the up regulation of CR3 also play a role in this process. This study provides a new preventive and therapeutic option for respiratory infectious diseases and lays the theoretical basis for the development of PepO as an immunomodulation agent.

Nuclear Factor κB/MicroRNA-155 Upregulates the Expression Pattern of Cytokines in Regulating the Relapse of Chronic Sinusitis with Nasal Polyps and the Underlying Mechanism of Glucocorticoid.

BackgroundThe aim of this study was to explore the upregulated nuclear factor κB (NF-κB)/microRNA-155 (miR-155) in regulating inflammatory responses and relapse of chronic rhinosinusitis (CRS) with nasal polyps (NP), which underlies the molecular mechanism of glucocorticoid treatment.Material/MethodsThe study recruited 25 patients with eosinophilic (Eos) CRSwNP, 25 patients with Non-Eos CRSwNP, 25 patients with CRS without NP (CRSsNP) and 30 patients with nasal septum deviation (control group). The expression of NF-κB/miR-155 and inflammatory cytokines was detected in epithelial tissue specimens. Additionally, a mouse model of Eos CRSwNP was established, and the mice were treated by NF-κB inhibitor, miR-155 antagomir, or dexamethasone (DEX) to explore the role of NF-κB/miR-155 and the anti-inflammatory effects of glucocorticoid treatment.ResultsResults showed that the expression level of NF-κB/miR-155 was significantly elevated in the Eos CRSwNP group, accompanied by the upregulation of cytokines: tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-4, IL-5 (P<0.05) compared with the control group, the CRSsNP group or the Non-Eos CRSwNP group. The upregulation of NF-κB/miR-155 increased inflammatory mediator cyclooxygenase2 (COX2) while decreasing anti-inflammatory mediator Src homology-2 domain-containing inositol 5-phosphatase 1 (SOCS1), which resulted in the aberrant expression pattern of cytokines in the mice model. DEX treatment inhibited the expression of cytokines and decreased the relapse rate of Eos CRSwNP via inhibiting NF-κB/miR-155 (P<0.05).ConclusionsThe upregulation of NF-κB/miR-155 was crucial in mediating the aberrant expression of inflammatory cytokines in Eos CRSwNP. This molecular mechanism is a concern with the high relapse rate of Eos CRSwNP. However, glucocorticoid treatment inhibited the relapse of CRSwNP via downregulation of NF-κB/miR-155.

SHIP interacts with adaptor protein Nck and restricts actin turnover in B cells

SH2 domain-containing inositol 5′-phosphatase (SHIP) has critical functions in regulating signal transduction. In additional to its lipid phosphatase activity, SHIP engages in multiple protein-protein interactions, which can serve to localize either SHIP or its binding partners to a particular subcellular domain. Knock-out and knock-down studies have elucidated that SHIP negatively regulates the accumulation of F-actin in leukocytes, usually resulting in inhibition of actin dependent cellular activities such as spreading and migration. Here, we demonstrate that overexpression of SHIP inhibits B cell antigen receptor (BCR)-mediated cell spreading in murine and human B cell lines. B cell stimulation via the BCR or pervanadate induces an interaction between SHIP and Nck, an adaptor protein known to promote actin polymerization. Using a fluorescence recovery after photobleaching (FRAP) assay, we demonstrate that overexpression of SHIP slows F-actin dynamics in BCR-stimulated B cells and this can be overcome by co-overexpression of Nck. Our data supports a role for SHIP in limiting actin turnover and suggests it may do so in part by sequestering Nck.

Fli-1 Activation through Targeted Promoter Activity Regulation Using a Novel 3', 5'-diprenylated Chalcone Inhibits Growth and Metastasis of Prostate Cancer Cells.

The friend leukemia integration 1 (Fli-1) gene is involved in the expression control of key genes in multiple pathogenic/physiological processes, including cell growth, differentiation, and apoptosis; this implies that Fli-1 is a strong candidate for drug development. In our previous study, a 3′,5′-diprenylated chalcone, (E)-1-(2-hydroxy-4-methoxy-3,5-diprenyl) phenyl-3-(3-pyridinyl)-propene-1-one (C10), was identified as a novel anti-prostate cancer (PCa) agent. Here, we investigated the molecular mechanisms underlying the anti-cancer effects of C10 on the growth, metastasis, and invasion of PC3 cells in vitro. Our results show that C10 exhibited a strong inhibitory effect on proliferation and metastasis of PC3 cells via several cellular and flow cytometric analyses. Further mechanism studies revealed that C10 likely serves as an Fli-1 agonist for regulating the expression of Fli-1 target genes including phosphatidylinositol 3-kinase (P110), murine double minute2 (MDM2), B-cell lymphoma-2 (Bcl-2), Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP-1), and globin transcription factor-1 (Gata-1) as well as the phosphorylation of extracellular-regulated protein kinases 1 (ERK1). Further, we confirmed that C10 can regulate the expressions of vascular endothelial growth factor 1 (VEGF-1), transforming growth factor-β2 (TGF-β2), intercellular cell adhesion molecule-1 (ICAM-1), p53, and matrix metalloproteinase 1 (MMP-1) genes associated with tumor apoptosis, migration, and invasion. Thus, C10 exhibits stronger anticancer activity with novel molecular targets and regulatory molecular mechanisms, indicating its great potency for development as a novel targeted anticancer drug.

Downregulation of SHIP2 by Hepatitis B Virus X Promotes the Metastasis and Chemoresistance of Hepatocellular Carcinoma through SKP2.

Hepatitis B virus (HBV)-encoded X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). The protein SH2 domain containing inositol 5-phosphatase 2 (SHIP2) belongs to the family of enzymes that dephosphorylate the 5 position of PI(3,4,5)P3 to produce PI(3,4)P2. Expression of SHIP2 has been associated with several cancers including HCC. However, its role in the development of HBV-related HCC remains elusive. In this study, we performed tissue microarray analysis using 49 cases of HCC to explore SHIP2 expression changes and found that SHIP2 was downregulated in HBV-positive HCC. In addition, S-phase kinase-associated protein 2 (SKP2), a component of the E3 ubiquitin–ligase complex, was increased in HCC cell lines that overexpressed HBx, which also showed a notable accumulation of polyubiquitinated SHIP2. Moreover, HCC cells with silenced SHIP2 had increased expression of mesenchymal markers, which promotes cell migration, enhances glucose uptake, and leads to resistance to the chemotherapy drug (5-Fluorouracil, 5-FU). Taken together, our results demonstrate that HBx downregulates SHIP2 through SKP2 and suggest a potential role for SHIP2 in HBx-mediated HCC migration.

MicroRNA 155 Contributes to Host Immunity against Leishmania donovani but Is Not Essential for Resolution of Infection.

CD4+ T helper 1 (Th1) cells producing interferon gamma (IFN-γ) are critical for the resolution of visceral leishmaniasis (VL). MicroRNA 155 (miR155) promotes CD4+ Th1 responses and IFN-γ production by targeting suppressor of cytokine signaling-1 (SOCS1) and Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP-1) and therefore could play a role in the resolution of VL. To determine the role of miR155 in VL, we monitored the course of Leishmania donovani infection in miR155 knockout (miR155KO) and wild-type (WT) C57BL/6 mice. miR155KO mice displayed significantly higher liver and spleen parasite loads than WT controls and showed impaired hepatic granuloma formation. However, parasite growth eventually declined in miR155KO mice, suggesting the induction of a compensatory miR155-independent antileishmanial pathway. Leishmania antigen-stimulated splenocytes from miR155KO mice produced significantly lower levels of Th1-associated IFN-γ than controls. Interestingly, at later time points, levels of Th2-associated interleukin-4 (IL-4) and IL-10 were also lower in miR155KO splenocyte supernatants than in WT mice. On the other hand, miR155KO mice displayed significantly higher levels of IFN-γ, iNOS, and TNF-α gene transcripts in their livers than WT mice, indicating that distinct organ-specific antiparasitic mechanisms were involved in control of L. donovani infection in miR155KO mice. Throughout the course of infection, organs of miR155KO mice showed significantly more PDL1-expressing Ly6Chi inflammatory monocytes than WT mice. Conversely, blockade of Ly6Chi inflammatory monocyte recruitment in miR155KO mice significantly reduced parasitic loads, indicating that these cells contributed to disease susceptibility. In conclusion, we found that miR155 contributes to the control of L. donovani but is not essential for infection resolution.

Solution structure of SHIP2 SH2 domain and its interaction with a phosphotyrosine peptide from c-MET

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) binds with the Y1356-phosphorylated hepatocyte growth factor (HGF) receptor, c-MET, through its SH2 domain, which is essential for the role of SHIP2 in HGF-induced cell scattering and cell spreading. Previously, the experimental structure of the SH2 domain from SHIP2 (SHIP2-SH2) had not been reported, and its interaction with the Y1356-phosphorylated c-MET had not been investigated from a structural point of view. In this study, the solution structure of SHIP2-SH2 was determined by NMR spectroscopy, where it was found to adopt a typical SH2-domain fold that contains a positively-charged pocket for binding to phosphotyrosine (pY). The interaction between SHIP2-SH2 and a pY-containing peptide from c-MET (Y1356 phosphorylated) was investigated through NMR titrations. The results showed that the binding affinity of SHIP2-SH2 with the phosphopeptide is at low micromolar level, and the binding interface consists of the positively-charged pocket and its surrounding regions. Furthermore, R28, S49 and R70 were identified as key residues for the binding and may directly interact with the pY. Taken together, these findings provide structural insights into the binding of SHIP2-SH2 with the Y1356-phosphorylated c-MET, and lay a foundation for further studies of the interactions between SHIP2-SH2 and its various binding partners.

Molecular analysis of oncogene expressions in different grades of gliomas

The aggressiveness of brain tumors is attributed to the expression of multiple oncogenes involved in proliferation, metabolism and therapeutic resistance whose potential correlation with tumor progression has not been well-studied. In this study, we aimed to investigate the relationship of oncotargets involved in pathogenesis with respect to glioma grades. Gliomas ( n =40) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing for the detection of epidermal growth factor receptor ( EGFR ) mutants. Expressions levels of EGFR , EGFR variant III ( EGFRvIII ), Lck/Yes novel tyrosine kinase ( LYN ), Spleen tyrosine kinase ( SYK ), insulin receptor substrate 1 ( IRS1 ), phosphatidylinositol 3-kinase ( PI3K ), Src homology 2 domain-containing inositol 5-phosphatase 1 ( SHIP1 ) and glucose transporter 3 ( GLUT3 ) were studied using real-time PCR and compared against glioma grades via statistical methods. Protein expressions were analyzed using immunohistochemistry and western blotting. EGFRvIII was detected in 53% and exon 4 deletion ( de4 EGFR ) in 20% of gliomas. Importantly, the expressions levels of candidate oncogenes were significantly upregulated ( P

Acute downregulation of miR-155 leads to a reduced collagen synthesis through attenuating macrophages inflammatory factor secretion by targeting SHIP1.

Fibrosis, tightly associated with fibroblasts collagen synthesis, is related closely with inflammatory response. Our previously study found that acute downregulation of miR-155 at wound sites leads to a reduced fibrosis, however its particular mechanism is unclear. Herein, we aimed to explore the mechanism of miR-155 in reducing fibrosis. We first found that down-regulation of miR-155 inhibited macrophages transforming growth factor-β1 (TGF-β1) and IL-1β secretion. Next, we found that co-cultured with macrophages increased the proliferation and collagen synthesis of fibroblasts, and downregulation of miR-155 in macrophages could effectively attenuate the accelerative effects. We further identified SH2 domain containing inositol-5-phosphatase 1 (SHIP1) as a direct target of miR-155 in macrophages, and the expression of SHIP1 was negatively correlated with the level of miR-155. We further confirmed that PI3K/Akt pathway was involved in this process. Last, we found that downregulation of miR-155 leads to a reduced fibrosis in sever burn rat. Taken together, these results indicate that down-regulation of miR-155 leads to a reduced fibroblasts proliferation and collagen synthesis through attenuating macrophages TGF-β1 and IL-1β secretion by targeting SHIP1 via PI3K/Akt pathway, suggesting its potential therapeutic effects on the treatment of skin fibrosis.