Domain-containing Inositol 5-phosphatase Research Articles

MiR-155–regulated molecular network orchestrates cell fate in the innate and adaptive immune response to Mycobacterium tuberculosis

The regulation of host-pathogen interactions during Mycobacterium tuberculosis (Mtb) infection remains unresolved. MicroRNAs (miRNAs) are important regulators of the immune system, and so we used a systems biology approach to construct an miRNA regulatory network activated in macrophages during Mtb infection. Our network comprises 77 putative miRNAs that are associated with temporal gene expression signatures in macrophages early after Mtb infection. In this study, we demonstrate a dual role for one of these regulators, miR-155. On the one hand, miR-155 maintains the survival of Mtb-infected macrophages, thereby providing a niche favoring bacterial replication; on the other hand, miR-155 promotes the survival and function of Mtb-specific T cells, enabling an effective adaptive immune response. MiR-155-induced cell survival is mediated through the SH2 domain-containing inositol 5-phosphatase 1 (SHIP1)/protein kinase B (Akt) pathway. Thus, dual regulation of the same cell survival pathway in innate and adaptive immune cells leads to vastly different outcomes with respect to bacterial containment.

IL-10/microRNA-155/SHIP-1 signaling pathway is crucial for commensal bacteria induced spontaneous colitis

Interleukin 10 (IL-10) microRNA-155 (miR-155)/Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP-1) signaling pathway plays an important role in maintaining immune homeostasis. We aimed to determine and characterize the changes induced by commensal bacteria on the IL-10/miR-155/SHIP-1 signaling pathway, as well as the potential therapeutic effects of anti-miR-155 on colitis in IL-10 deficient (IL-10−/−) mice. Age- and sex-matched C57BL/6 IL-10−/− and wild type mice were transferred from a germ-free environment to a specific pathogen free condition. Part of IL-10−/− mice were then treated with anti-miR-155. IL-10/miR-155/SHIP-1 signaling pathway was evaluated and the therapeutic effects of anti-miR-155 treatment on colitis in IL-10−/− mice was assessed. The expression and the relationship of IL-10, miR-155, and SHIP-1 were also measured in patients with active Crohn’s colitis. IL-10/miR-155/SHIP-1 signaling pathway was activated in IL-10−/− mice transferring from a germ-free environment to a specific pathogen free condition. Anti-miR-155 treatment significantly ameliorated the severity of colitis in IL-10−/− mice. Additionally, administration of anti-miR-155 was associated with a restoration of SHIP-1 signaling pathway. The relationship of IL-10, miR-155, and SHIP-1 was confirmed in human study using samples from patients with active Crohn’s colitis. IL-10/miR-155/SHIP-1 pathways play a critical role in commensal bacteria induced colitis and miR-155 may be a potential therapeutic target for human inflammatory bowel disease.

Human Cytomegalovirus Induces an Atypical Activation of Akt To Stimulate the Survival of Short-Lived Monocytes.

Human cytomegalovirus (HCMV) is a pervasive herpesvirus responsible for significant morbidity and mortality among immunodeficient/naive hosts. Following a primary HCMV infection, circulating blood monocytes mediate the systemic spread of the virus. Extending the short 48-h life span of monocytes is critical to the viral dissemination process, as these blood-borne cells are nonpermissive for virus replication until they are fully differentiated into macrophages. Here, we show that HCMV glycoprotein gB binding to cellular epidermal growth factor receptor (EGFR) during HCMV entry initiated a rapid (within 15 min) activation of the apoptosis suppressor Akt, which was maintained through 72 h. The virus-induced activation of Akt was more robust than that with the normal myeloid growth factor macrophage colony-stimulating factor (M-CSF) and was essential for infected monocytes to bypass the 48-h viability checkpoint. Activation of phosphoinositide 3-kinase (PI3K) following EGFR engagement by HCMV mediated the phosphorylation of Akt. Moreover, HCMV entry drove a switch away from the PI3K p110δ isoform, which was required for the viability of uninfected monocytes, to the p110β isoform in order to facilitate the Akt-dependent prosurvival state within infected cells. Simultaneously, in contrast to M-CSF, HCMV promoted a rapid increase in SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) expression, leading to signaling through a noncanonical Akt activation pathway. To ensure maximum Akt activity, HCMV also induced an early phosphorylation-dependent inactivation of the negative regulator phosphatase and tensin homolog. Overall, our data indicate that HCMV hijacks the upstream Akt signaling network to induce a nontraditional activation of Akt and subsequently a prosurvival decision at the 48-h cell fate checkpoint, a vital step for HCMV's dissemination and persistence strategy. HCMV is found throughout the world with a prevalence of 55 to 100% within the human population. HCMV infection is generally asymptomatic in immunocompetent or naive individuals but is a significant cause of morbidity and mortality among the immunocompromised. Widespread organ inflammation is associated with symptomatic infections, which is a direct consequence of the viral dissemination strategy. Inflammatory peripheral blood monocytes facilitate the spread of HCMV. However, HCMV must subvert the naturally short life span of monocytes. In this work, we demonstrate that HCMV induces the activation of Akt, an antiapoptotic protein, in a manner distinct from that of normal myeloid growth factors. Moreover, we decipher how HCMV dysregulates the upstream Akt signaling network during viral entry to promote an Akt-dependent prosurvival state following infection. Delineation of the virus-specific mechanisms that regulate cellular prosurvival pathways in order to drive the survival of HCMV-infected monocytes is important to identifying new anti-HCMV therapeutic targets.

SH2 domain-containing inositol 5-phosphatase (SHIP2) inhibition ameliorates high glucose-induced de-novo lipogenesis and VLDL production through regulating AMPK/mTOR/SREBP1 pathway and ROS production in HepG2 cells

Hepatic de-novo lipogenesis and production of triglyceride rich very low density lipoprotein (VLDL) is increased in the state of insulin resistance, however, the role of a negative regulator of the insulin signaling pathway, the SH2 domain-containing inositol 5-phosphatase (SHIP2) in this process, remains unknown. In the present study, we studied the molecular mechanisms linking SHIP2 expression to metabolic dyslipidemia using overexpression or suppression of SHIP2 gene in HepG2 cells exposed to high glucose (33mM). The results showed that high glucose induced SHIP2 mRNA and protein levels in HepG2 cells. Overexpression of the dominant negative mutant SHIP2 (SHIP2-DN) ameliorated high glucose-induced de-novo lipogenesis and secretion of apoB containing lipoprotein in HepG2 cells, as demonstrated by a reduction in both secreted apoB and MTP expression, and decreased triglyceride levels and the expression of lipogenic genes such as SREBP1c, FAS and ACC. Overexpression of the SHIP2-DN decreased high glucose-induced apoB containing lipoproteins secretion via reduction in ROS generation, JNK phosphorylation and Akt activation. Furthermore, using the specific inhibitor and activator, it was found that the AMPK/mTOR/SREBP1 is the signaling pathway that mediates the effects of SHIP2 modulation on hepatic de-novo lipogenesis. Taken together, these findings suggest that SHIP2 is an important regulator of hepatic lipogenesis and lipoprotein secretion in insulin resistance state.

SH2 domain-containing inositol 5-phosphatase (SHIP2) regulates de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells

Hepatic de-novo lipogenesis and production of triglyceride rich VLDL are regulated via the phosphoinositide 3-kinase cascade, however, the role of a negative regulator of this pathway, the SH2 domain-containing inositol 5-phosphatase (SHIP2) in this process, remains unknown. In the present study, we investigated the molecular link between SHIP2 expression and metabolic dyslipidemia using overexpression or suppression of SHIP2 gene in HepG2 cells. The results showed that overexpression of the wild type SHIP2 gene (SHIP2-WT) led to a higher total lipid content (28%) compared to control, whereas overexpression of the dominant negative SHIP2 gene (SHIP2-DN) reduced total lipid content in oleate treated cells by 40%. Overexpression of SHIP2-WT also led to a significant increase in both secretion of apoB100 containing lipoproteins and de-novo lipogenesis, as demonstrated by an enhancement in secreted apoB100 and MTP expression, increased intra and extracellular triglyceride levels and enhanced expression of lipogenic genes such as SREBP1c, FAS and ACC. On the other hand, overexpression of the SHIP2-DN gene prevented oleate-induced de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells. Collectively, these findings suggest that SHIP2 expression level is a key determinant of hepatic lipogenesis and lipoprotein secretion, and its inhibition could be considered as a potential target for treatment of dyslipidemia.

CRISPR/CAS9-Mediated Genome Editing of miRNA-155 Inhibits Proinflammatory Cytokine Production by RAW264.7 Cells.

MicroRNA 155 (miR-155) is a key proinflammatory regulator in clinical and experimental rheumatoid arthritis (RA). Here we generated a miR-155 genome knockout (GKO) RAW264.7 macrophage cell line using the clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CAS9) technology. While upregulating the Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1), the miR-155 GKO line is severely impaired in producing proinflammatory cytokines but slightly increased in osteoclastogenesis upon treatment with receptor activator of nuclear factor-κB ligand (RANKL). Taken together, our results suggest that genome editing of miR-155 holds the potential as a therapeutic strategy in RA.

Immune cell inhibition by SLAMF7 is mediated by a mechanism requiring src kinases, CD45, and SHIP-1 that is defective in multiple myeloma cells.

Signaling lymphocytic activation molecule F7 (SLAMF7) is a receptor present on immune cells, including natural killer (NK) cells. It is also expressed on multiple myeloma (MM) cells. This led to development of an anti-SLAMF7 antibody, elotuzumab, showing efficacy against MM. SLAMF7 mediates activating or inhibitory effects in NK cells, depending on whether cells express or do not express the adaptor EAT-2. Since MM cells lack EAT-2, we elucidated the inhibitory effectors of SLAMF7 in EAT-2-negative NK cells and tested whether these effectors were triggered in MM cells. SLAMF7-mediated inhibition in NK cells lacking EAT-2 was mediated by SH2 domain-containing inositol phosphatase 1 (SHIP-1), which was recruited via tyrosine 261 of SLAMF7. Coupling of SLAMF7 to SHIP-1 required Src kinases, which phosphorylated SLAMF7. Although MM cells lack EAT-2, elotuzumab did not induce inhibitory signals in these cells. This was at least partly due to a lack of CD45, a phosphatase required for Src kinase activation. A defect in SLAMF7 function was also observed in CD45-deficient NK cells. Hence, SLAMF7-triggered inhibition is mediated by a mechanism involving Src kinases, CD45, and SHIP-1 that is defective in MM cells. This defect might explain why elotuzumab eliminates MM cells by an indirect mechanism involving the activation of NK cells.

MicroRNA-155 influences B-cell receptor signaling and associates with aggressive disease in chronic lymphocytic leukemia

AbstractHigh-level leukemia cell expression of micro-RNA 155 (miR-155) is associated with more aggressive disease in patients with chronic lymphocytic leukemia (CLL), including those cases with a low-level expression of ζ-chain–associated protein of 70 kD. CLL with high-level miR-155 expressed lower levels of Src homology-2 domain-containing inositol 5-phosphatase 1 and were more responsive to B-cell receptor (BCR) ligation than CLL with low-level miR-155. Transfection with miR-155 enhanced responsiveness to BCR ligation, whereas transfection with a miR-155 inhibitor had the opposite effect. CLL in lymphoid tissue expressed higher levels of miR155HG than CLL in the blood of the same patient. Also, isolated CD5brightCXCR4dim cells, representing CLL that had been newly released from the microenvironment, expressed higher levels of miR-155 and were more responsive to BCR ligation than isolated CD5dimCXCR4bright cells of the same patient. Treatment of CLL or normal B cells with CD40-ligand or B-cell–activating factor upregulated miR-155 and enhanced sensitivity to BCR ligation, effects that could be blocked by inhibitors to miR-155. This study demonstrates that the sensitivity to BCR ligation can be enhanced by high-level expression of miR-155, which in turn can be induced by crosstalk within the tissue microenvironment, potentially contributing to its association with adverse clinical outcome in patients with CLL.

SHIP2: A “NEW” Insulin Pathway Target for Aging Research

Strong evidence suggests that systemic inflammation and central adiposity contribute to and perpetuate metabolic syndrome. All of these alterations predispose individuals to type 2 diabetes mellitus (T2DM), cardiovascular disease, as well as Alzheimer's disease (AD), all characterized by chronic inflammatory status. On the other hand, extensive abnormalities in insulin and insulin-like growth factor I (IGF-I) and IGF-II signaling mechanisms in brains with AD have been demonstrated, suggesting that AD could be a third form of diabetes. The Src homology domain-containing inositol 5-phosphatase 2 (SHIP2) has an important role in the insulin pathway because its over-expression causes impairment of insulin/IGF-1 signaling. Because some single-nucleotide polymorphisms (SNP) of the gene encoding SHIP2 were significantly associated in T2DM patients with metabolic syndrome and some related conditions, we decided to conduct a case-control study on this gene, analyzing AD and T2DM subjects as cases and young, old, and centenarians as controls. Our results suggest a putative correlation between the the rs144989913 SNP and aging, both successful and unsuccessful, rather than age-related diseases. Because this SNP is an insertion/deletion of 28 bp, it might cause an alteration in SHIP2 expression. It is noteworthy that SHIP2 has been demonstrated to be a potent negative regulator of insulin signaling and insulin sensitivity. Many studies demonstrated the association of the insulin/IGF1 pathway with aging and longevity, so it is tempting to speculate that the found association with SHIP2 and aging might depend on its effect on the insulin/IGF-1 pathway.

Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants

Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

Human Mesenchymal Stem Cells Suppress the Stretch–Induced Inflammatory miR-155 and Cytokines in Bronchial Epithelial Cells

Current research in pulmonary pathology has focused on inflammatory reactions initiated by immunological responses to allergens and irritants. In addition to these biochemical stimuli, physical forces also play an important role in regulating the structure, function, and metabolism of the lung. Hyperstretch of lung tissues can contribute to the inflammatory responses in asthma, but the mechanisms of mechanically induced inflammation in the lung remain unclear. Our results demonstrate that excessive stretch increased the secretion of inflammatory cytokines by human bronchial epithelial cells (hBECs), including IL-8. This increase of IL-8 secretion was due to an elevated microRNA-155 (miR-155) expression, which caused the suppression of Src homology 2 domain–containing inositol 5-phosphatase 1 (SHIP1) production and the subsequent activation of JNK signaling. In vivo studies in our asthmatic mouse model also showed such changes in miR-155, IL-8, and SHIP1 expressions that reflect inflammatory responses. Co-culture with human mesenchymal stem cells (hMSCs) reversed the stretch-induced hBEC inflammatory responses as a result of IL-10 secretion by hMSCs to down-regulate miR-155 expression in hBECs. In summary, we have demonstrated that mechanical stretch modulates the homeostasis of the hBEC secretome involving miR-155 and that hMSCs can be used as a potential therapeutic approach to reverse bronchial epithelial inflammation in asthma.

How does SHIP1/2 balance PtdIns(3,4)P2 and does it signal independently of its phosphatase activity?

The number of cellular events identified as being directly or indirectly modulated by phosphoinositides dramatically increased in the recent years. Part of the complexity results from the fact that the seven phosphoinositides play second messenger functions in many different areas of growth factors and insulin signaling, cytoskeletal organization, membrane dynamics, trafficking, or nuclear signaling. PtdIns(3,4)P2 is commonly reported as a product of the SH2 domain-containing inositol 5-phosphatases 1/2 (SHIP1 and SHIP2) that dephosphorylate PtdIns(3,4,5)P3 at the 5-position. Here we discuss recent interest in PtdIns(3,4)P2 signaling highlighting its involvement in key cellular mechanisms such as cell adhesion, migration, and cytoskeletal regulation. We question and discuss the involvement of SHIP2 either as a PI 5-phosphatase or as a scaffold protein in insulin signaling, cytoskeletal dynamics, and endocytosis of growth factor receptors.

House Dust Mite-Specific Immunotherapy Alters the Basal Expression of T Regulatory and FcεRI Pathway Genes

Background: Regulatory T (Treg) cells and IgE-mediated signaling pathways could play important roles in the induction of allergen tolerance during house dust mite-specific subcutaneous immunotherapy (HDM-SCIT). Our aim was to compare the basal expression levels of Treg, T helper 1 (Th1) and Th2 transcription factors and components involved in IgE-mediated signaling in healthy subjects with those in HDM-allergic patients both untreated and successfully treated with HDM-SCIT. Methods: Thirty-nine HDM-allergic patients who completed a 3- to 5-year course of mite extract SCIT, 20 mite-allergic controls and 25 healthy controls participated in this study. The efficacy of SCIT was monitored using skin-prick tests (SPTs), total immunoglobulin E (tIgE), specific IgE (sIgE), sIgG₄, nasal challenge and visual analog scale (VAS) scores at several time points. The mRNA levels of forkhead box protein 3 (FOXP3), T-BET, GATA-3, FcεRI, spleen tyrosine kinase (Syk), phosphatidylinositol 3 kinase (PI3K) and SH2 domain-containing inositol phosphatase (SHIP) were quantified by real-time RT-PCR using nonstimulated whole blood samples. Results: Decreased wheal sizes and VAS scores, negative challenges and increased sIgG₄ levels indicated that SCIT was effective in the treated patients. Basal expression levels of FOXP3 and GATA-3 decreased and T-BET levels increased in both treated patients and in healthy controls compared to untreated patients. The IgE-mediated pathway kinases Syk and PI3K exhibited reduced expression, whereas SHIP phosphatase levels were elevated in both treated patients and healthy controls relative to untreated patients. The expression levels of FcεRI were not significantly altered. Conclusions: Immunotherapy using HDM extracts results in a modification of the basal expression levels of several IgE-related signaling factors and induces a highly significant upregulation of Th1-response and downregulation of Th2-response transcription factors. Interestingly, this therapy also appears to reduce the basal expression of FOXP3.

Reversible Ser/Thr SHIP phosphorylation: A new paradigm in phosphoinositide signalling?

Phosphoinositide (PI) phosphatases such as the SH2 domain-containing inositol 5-phosphatases 1/2 (SHIP1 and 2) are important signalling enzymes in human physiopathology. SHIP1/2 interact with a large number of immune and growth factor receptors. Tyrosine phosphorylation of SHIP1/2 has been considered to be the determining regulatory modification. However, here we present a hypothesis, based on recent key publications, highlighting the determining role of Ser/Thr phosphorylation in regulating several key properties of SHIP1/2. Since a subunit of the Ser/Thr phosphatase PP2A has been shown to interact with SHIP2, a putative mechanism for reversing SHIP2 Ser/Thr phosphorylation can be anticipated. PI phosphatases are potential target molecules in human diseases, particularly, but not exclusively, in cancer and diabetes. Therefore, this novel regulatory mechanism deserves further attention in the hunt for discovering novel or complementary therapeutic strategies. This mechanism may be more broadly involved in regulating PI signalling in the case of synaptojanin1 or the phosphatase, tensin homolog, deleted on chromosome TEN.

Can Alzheimer Disease Be a Form of Type 3 Diabetes?

Alzheimer disease (AD) and metabolic syndrome are two highly prevalent pathological conditions of Western society due to incorrect diet, lifestyle, and vascular risk factors. Recent data have suggested metabolic syndrome as an independent risk factor for AD and pre-AD syndrome. Furthermore, biological plausibility for this relationship has been framed within the "metabolic cognitive syndrome" concept. Due to the increasing aging of populations, prevalence of AD in Western industrialized countries will rise in the near future. Thus, new knowledge in the area of molecular biology and epigenetics will probably help to make an early molecular diagnosis of dementia. An association between metabolic syndrome and specific single-nucleotide polymorphisms (SNPs) in the gene INPPL1, encoding for SHIP2, a SH2 domain-containing inositol 5-phosphatase involved in insulin signaling, has been described. According to recent data suggesting that Type 2 diabetes represents an independent risk factor for AD and pre-AD, preliminary results of a case-control study performed to test the putative association between three SNPs in the SHIP2 gene and AD show a trend toward association of these SNPs with AD.

Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells

S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.

Evidence of SHIP2 Ser132 phosphorylation, its nuclear localization and stability

PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are major signalling molecules in mammalian cell biology. PtdIns(3,4)P2 can be produced by PI3Ks [PI (phosphoinositide) 3-kinases], but also by PI 5-phosphatases including SHIP2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2]. Proteomic studies in human cells revealed that SHIP2 can be phosphorylated at more than 20 sites, but their individual function is unknown. In a model of PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null astrocytoma cells, lowering SHIP2 expression leads to increased PtdIns(3,4,5)P3 levels and Akt phosphorylation. MS analysis identified SHIP2 phosphosites on Ser132, Thr1254 and Ser1258; phosphotyrosine-containing sites were undetectable. By immunostaining, total SHIP2 concentrated in the perinuclear area and in the nucleus, whereas SHIP2 phosphorylated on Ser132 was in the cytoplasm, the nucleus and nuclear speckles, depending on the cell cycle stage. SHIP2 phosphorylated on Ser132 demonstrated PtdIns(4,5)P2 phosphatase activity. Endogenous phospho-SHIP2 (Ser132) showed an overlap with PtdIns(4,5)P2 staining in nuclear speckles. SHIP2 S132A was less sensitive to C-terminal degradation and more resistant to calpain as compared with wild-type enzyme. We have identified nuclear lamin A/C as a novel SHIP2 interactor. We suggest that the function of SHIP2 is different at the plasma membrane where it recognizes PtdIns(3,4,5)P3, and in the nucleus where it may interact with PtdIns(4,5)P2, particularly in speckles.

Fc gamma receptor IIb modulates the molecular Grb2 interaction network in activated B cells

B cells require signals transduced by the B cell antigen receptor (BCR) to provide humoral adaptive immunity. These signals are modulated by co-receptors like the Fcγ receptor IIb (FcγRIIb) that prevents activation of B cells after co-ligation with the BCR. Positive and negative effectors need to be precisely organized into signaling complexes, which requires adapter proteins like the growth factor receptor-bound protein 2 (Grb2). Here, we address the question how Grb2-mediated signal integration is affected by FcγRIIb. Our data reveal that concomitant engagement of BCR and FcγRIIb leads to markedly increased Grb2-mediated formation of ternary protein complexes comprising downstream of kinase-3 (Dok-3), Grb2, and the SH2 domain-containing inositol phosphatase (SHIP). Consistently, we found Grb2 to be required for full FcγRIIb-mediated negative regulation. To investigate how FcγRIIb influences the entire Grb2 interactions, we utilized quantitative mass spectrometry to make a differential interactome analysis. This approach revealed a shift of Grb2 interactions towards negative regulators like Dok-3, SHIP and SHP-2 and reduced binding to other proteins like CD19. Hence, we provide evidence that Grb2-mediated signal integration is a dynamic process that is important for the crosstalk between the BCR and its co-receptor FcγRIIb.

IL-6 increases B-cell IgG production in a feed-forward proinflammatory mechanism to skew hematopoiesis and elevate myeloid production

AbstractSrc homology 2 domain-containing inositol 5-phosphatase (SHIP−/−) animals display an age-related increase in interleukin-6 (IL-6), a decrease in B lymphopoiesis, and an elevation in myelopoiesis. We investigated the origin of the IL-6 production and show that it is largely produced by peritoneal and splenic macrophages. IL-6 production by these macrophages is not a direct result of the loss of SHIP: IL-6 production is not spontaneous, is absent from bone marrow-derived macrophages, declines with prolonged culture of macrophages, and requires a stimulus present in vivo. The IL-6–rich peritoneal cavity of SHIP−/− mice shows more than 700-fold more immunoglobulin G (IgG) than wild-type, approximately 20% of which is aggregated or in an immune complex and contains B220+ cells that secrete IgG. The SHIP-deficient peritoneal macrophages show evidence of IgG receptor stimulation. Animals lacking both the signal-transducing γ-chain of IgG receptors and SHIP or Ig and SHIP produce less IL-6. The data indicate a feed-forward process in which peripheral macrophages, responding through IgG receptors to secreted IgG, produce IL-6, to support further B-cell production of IgG. Because of the proinflammatory phenotype of SHIP−/− animals, these findings emphasize the importance of IL-6–neutralizing strategies in autoimmune and proinflammatory diseases.

Abstract 3005: Significant roles of aberrant miRNA expressions in NK-cell lymphomagenesis

Abstract Background: Natural Killer (NK) cell lymphomas/leukemias are characterized groups of highly aggressive lymphoid malignancies, which are comprised of “extranodal NK/T cell lymphoma, nasal type” and “aggressive NK-cell leukemia”. Notably, these two subtypes show many similarities in their morphologic features, immmunophenotypes and genotypes, and are invariably associated with Epstein-Barr virus (EBV), which suggests they may share the same genetic alterations. The gene(s) responsible for natural killer (NK)-cell lymphoma/leukemia have not been identified. Materials and Methods: In the present study, we used miRNA array, Northern and quantitative PCR analyses to screen for and quantitatively assess miRNA expression in NK-cell lymphomas/leukemias using 70 probe sets of miRNAs. For function analysis, we conducted antisense oligonucleotides and lentivirus transfection assays using NK-cell lymphoma cell lines. Results: We found that in NK-cell lymphoma lines (n=10) and specimens of primary lymphoma (n=10), levels of miR-21 and miR-155 expression were significantly higher than those seen in normal natural killer (CD3-CD56+) cells (n=8). Furthermore, miR-150 expression of primary samples and cell lines was significantly lower than CD3-CD56+ normal cells. To determine the functions of these microRNAs in lymphomagenesis, we examined the effects of antisense oligonucleotides targeting miR-21 (ASO-21) and/or miR-155 (ASO-155) in NK-cell lymphoma lines overexpressing one or both of these miRNAs. Conversely, cells showing little endogenous expression of miR-21, miR-155 and miR-150 were transduced using lentiviral vectors, leading to their overexpression. Reducing expression of miR-21 or miR-155 led to upregulation of phosphatase and tensin homologue (PTEN), programmed cell death 4 (PDCD4), or Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1). ASO-21- and ASO-155-treated cell lines all showed downregulation of phosphorylated AKTser473 (pAKT). Moreover, transduction with either miR-21 or miR-155 led to downregulation of PTEN and PDCD4, or SHIP1 with upregulation of pAKT. Because apoptosis and cell cycle arrest were induced by transduction of miR-150 into NK-cell lymphoma cell lines, the miRNA is possible candidate of tumor suppressive miRNA in this tumor entity. Detection for direct target of the miR-150 is now underway. Conclusions: Because recent reports suggest that EBV infection can lead directly to upregulation of miR-21 and miR-155, aberrant overexpressions of these miRNAs via EBV infection could be the first-hit genetic alterations during NK-cell lymphomagenesis. Collectively, these results provide important new insight into the pathogenesis of NK-cell lymphoma/leukemia and suggest targeting these miRNAs may represent a useful approach to treating NK-cell lymphoma/leukemia. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3005.