Because phosphoinositide 3-kinase (PI3K) plays a central role in cellular activation, proliferation, and survival, pharmacologic inhibitors targeting components of the PI3K pathway are actively being developed as therapeutics for the treatment of inflammatory disorders and cancer. These targeted drugs inhibit the activity of either PI3K itself or downstream protein kinases. However, a previously unexplored, alternate strategy is to activate the negative regulatory phosphatases in this pathway. The SH2-containing inositol-5-phosphatase SHIP1 is a normal physiologic counterregulator of PI3K in immune/hematopoietic cells that hydrolyzes the PI3K product phosphatidylinositiol-3,4,5-trisphosphate (PIP 3 ). We now describe the identification and characterization of potent and specific small-molecule activators of SHIP1. These compounds represent the first small-molecule activators of a phosphatase, and are able to activate recombinant SHIP1 enzyme in vitro and stimulate SHIP1 activity in intact macrophage and mast cells. Mechanism of activation studies with these compounds suggest that they bind a previously undescribed, allosteric activation domain within SHIP1. Furthermore, in vivo administration of these compounds was protective in mouse models of endotoxemia and acute cutaneous anaphylaxis, suggesting that SHIP1 agonists could be used therapeutically to inhibit the PI3K pathway. IntroductionIn response to extracellular signals, phosphoinositide 3-kinase (PI3K) becomes activated and phosphorylates phosphatidylinositol-4,5-bisphosphate (PI-4,5-P 2 ) within the plasma membrane to generate phosphatidylinositol-3,4,5-bisphosphate (PIP 3 ). PIP 3 then initiates a cascade of downstream signaling pathways by interacting with pleckstrin homology (PH) domain-containing proteins, such as protein kinase B (PKB, also known as Akt), that regulate cellular activation, proliferation and/or survival, depending on the cell type and stimulus. 1 Cellular levels of PIP 3 are normally tightly regulated by both PI3K and the 5Ј inositol phosphatases SHIP1 (SH2 domain-containing inositol phosphatase) and SHIP2, as well as the 3Ј inositol phosphatase PTEN, which dephosphorylates PIP 3 . 2,3 Of these, SHIP1 is unique in that its expression is restricted primarily to immune and hematopoietic cells. 2,4 SHIP's role in immune cell homeostasis is shown both by the myeloproliferative syndrome observed in SHIP1 Ϫ/Ϫ mice, as well as the hypersensitivity of SHIP1 Ϫ/Ϫ mice and cells to immune stimulation. 5,6 SHIP1 mediates signaling from the inhibitory Fc␥RIIB receptor, 7 and is important in terminating signal transduction from activating immune/hematopoietic cell receptor systems. 8 Diminished SHIP1 activity or expression has been observed in human inflammatory diseases 9 and hematopoietic malignancies. [10][11][12][13] Because dysregulated activation of the PI3K pathway contributes to inflammatory/immune disorders and cancer, intense efforts have been invested into the development of inhibitors of PI3K itself, as well as downstream protein kinas...

The cytokine interleukin-10 (IL-10) potently inhibits macrophage function through activation of the transcription factor STAT3. The expression of SOCS3 (suppressor of cytokine signaling-3) has been shown to be induced by IL-10 in a STAT3-dependent manner. However, the relevance of SOCS3 expression to the anti-inflammatory effect of IL-10 on macrophages has been controversial. Through kinetic analysis of the requirement for SOCS3 in IL-10 inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-␣ (TNF␣) transcription and translation, SOCS3 was found to be necessary for TNF␣ expression during the early phase, but not the late phase of IL-10 action. SOCS3 was essential for IL-10 inhibition of LPS-stimulated production of iNOS (inducible nitricoxide synthase) protein and nitric oxide (NO 204 and Tyr 221 were the only structural features of SOCS3 that were necessary in mediating IL-10 inhibition of iNOS protein expression and NO production. These data define SOCS3 as an important mediator of IL-10 inhibition of macrophage activation and that SOCS3 interferes with distinct LPS-stimulated signal transduction events through differing mechanisms.Macrophages form the first line of defense against foreign pathogens by producing pro-inflammatory mediators that either directly attack the invading organism, or recruit and activate other cells of the innate and acquired immune systems (1). Macrophage recognition of pathogens is mediated by pattern-recognition receptors, including the members of the Toll-like receptor family (TLR), 3 which recognize microbial structures forming pathogen-associated molecular patterns (2). A well characterized pathogen-associated molecular pattern is the cell wall component of Gram-negative bacteria called lipopolysaccharide (LPS).Binding of LPS to the TLR4 complex on macrophages leads to rapid macrophage activation. Whereas this inflammatory response is usually helpful and protective to the host, if left unchecked, the excessive amount of inflammatory cytokines produced leads to tissue destruction and pathologies such as septic shock (3).Interleukin-10 (IL-10) is a key physiological negative regulator of macrophage activation (4). Activated macrophages produce various inflammatory mediators including tumor necrosis factor-␣ (TNF␣) and nitric oxide (NO), all of which are suppressed by IL-10 (5, 6). Various animal model studies have substantiated the in vivo importance of an anti-inflammatory role for IL-10. Mice in which the IL-10 gene is deleted develop inflammatory bowel disease (7) and exaggerated immune reactions when challenged with antigen or LPS (8, 9). Administration of IL-10, however, ameliorates disease in models of endotoxemia (10), transplantation (11), and autoimmunity (12). In humans, the presence of elevated endogenous IL-10 is a positive prognostic variable in autoimmune disease (13) and allogeneic transplant patients (14).IL-10 initiates its actions on target cells by binding to the heterodimeric IL-10 receptor that belongs to the class II cytokine receptor famil...

The anti-inflammatory cytokine interleukin-10 (IL-10) is essential for attenuating the inflammatory response, which includes reducing the expression of pro-inflammatory microRNA-155 (miR-155) in lipopolysaccharide (LPS) activated macrophages. miR-155 enhances the expression of pro-inflammatory cytokines such as TNFα and suppresses expression of anti-inflammatory molecules such as SOCS1. Therefore, we examined the mechanism by which IL-10 inhibits miR-155. We found that IL-10 treatment did not affect the transcription of the miR-155 host gene nor the nuclear export of pre-miR-155, but rather destabilized both pri-miR-155 and pre-miR-155 transcripts, as well as interfered with the final maturation of miR-155. This inhibitory effect of IL-10 on miR-155 expression involved the contribution of both the STAT3 transcription factor and the phosphoinositol phosphatase SHIP1. This is the first report showing evidence that IL-10 regulates miRNA expression post-transcriptionally.