IL-3-mediated TNF production is necessary for mast cell development

Mouse mast cell development and survival are largely controlled by the cytokines IL-3 and stem cell factor (SCF). We have found that IL-3 stimulation of bone marrow cells induces the production of TNF via a PI3K- and MAPK kinase/ERK-dependent pathway. Specifically, Mac-1-positive cells were responsible for TNF production, which peaked on days 7-10 of culture and decreased rapidly thereafter. The importance of IL-3-induced TNF secretion was demonstrated by the failure of TNF-deficient bone marrow cells to survive for >3 wk when cultured in IL-3 and SCF, a defect that was reversed by the addition of soluble TNF. The development of human mast cells from bone marrow progenitors was similarly hampered by the addition of TNF-blocking Abs. Cell death was due to apoptosis, which occurred with changes in mitochondrial membrane potential and caspase activation. Apoptosis appeared to be due to loss of IL-3 signaling, because TNF-deficient cells were less responsive than their wild-type counterparts to IL-3-mediated survival. In vitro cultured mast cells from TNF-deficient mice also demonstrated reduced expression of the high affinity IgE receptor, which was restored to normal levels by the addition of soluble TNF. Finally, TNF-deficient mice demonstrated a 50% reduction in peritoneal mast cell numbers, indicating that TNF is an important mast cell survival factor both in vitro and in vivo.     

Regulating PLK1 dynamics by Cullin3/KLHL22-mediated ubiquitylation

FAS-associated factor 1, FAF1, is an evolutionarily conserved protein that has several protein interaction domains. Although FAF1 was initially identified as a member of the FAS death-inducing signaling complex, subsequent work has revealed that FAF1 functions in diverse biological processes. FAF1 has been shown to play an important role in normal development and neuronal cell survival, whereas FAF1 down regulation may contribute to multiple aspects of tumorigenesis. In particular, there is compelling evidence implicating FAF1 as a tumor suppressor involved in the regulation of apoptosis and NF-κB activity, as well as in ubiquitination and proteasomal degradation. Here, we highlight FAF1’s role in NF-κB signaling and postulate that this pathway has critical connotations for the pathogenesis and treatment of human cancers.     

IL-1 receptor-associated kinase 4 is essential for IL-18-mediated NK and Th1 cell responses

IL-18 is an important cytokine for both innate and adaptive immunity. NK T cells and Th1 cells depend on IL-18 for their divergent functions. The IL-18R, IL-1R, and mammalian Toll-like receptors (TLRs) share homologous intracellular domains known as the TLR/IL-1R/plant R domain. Previously, we reported that IL-1R-associated kinase (IRAK)-4 plays a critical role in IL-1R and TLR signaling cascades and is essential for the innate immune response. Because TLR/IL-1R/plant R-containing receptors mediate signal transduction in a similar fashion, we investigated the role of IRAK-4 in IL-18R signaling. In this study, we show that IL-18-induced responses such as NK cell activity, Th1 IFN-gamma production, and Th1 cell proliferation are severely impaired in IRAK-4-deficient mice. IRAK-4(-/-) Th1 cells also do not exhibit NF-kappaB activation or IkappaB degradation in response to IL-18. Moreover, AP-1 activation which is triggered by c-Jun N-terminal kinase activation is also completely inhibited in IRAK-4(-/-) Th1 cells. These results suggest that IRAK-4 is an essential component of the IL-18 signaling cascade.     

Doppel-induced Purkinje cell death is stoichiometrically abrogated by prion protein

Activin A can induce erythroid differentiation, whereas basic fibroblast growth factor (bFGF) can maintain the undifferentiated status of erythroid progenitors. How these two factors together can affect the regulation of erythroid differentiation in hematopoietic cells has not been elucidated. This study demonstrates that bFGF antagonizes activin A-mediated growth inhibition and hemoglobin (Hb) synthesis in K562 cells. Analyses of mitogen-activated protein kinases revealed that activin A-induced p38 phosphorylation and inhibited ERK1/2 phosphorylation. In contrast, bFGF worked antagonistically to induce ERK1/2 phosphorylation and inhibited p38 phosphorylation in K562 cells. Furthermore, co-treatment of cells with activin A and bFGF decreased p38 phosphorylation and increased ERK1/2 phosphorylation. SB203580 inhibition of p38 activity eliminated activin A-mediated growth inhibition and Hb synthesis, whereas U0126 inhibition of ERK1/2 activity augmented the effects of activin A on K562 cells. These results suggest that bFGF can negatively modulate p38 and positively modulate ERK1/2 to antagonize activin A-mediated growth inhibition and Hb synthesis in K562 cells.     

The role of the IL-22/IL-22R1 axis in cancer

Interleukin-22 (IL-22) is an IL-10 family cytokine produced by T cells and innate lymphoid cells. The IL-22 signaling pathway orchestrates mucosal immune defense and tissue regeneration through pleiotropic effects including pro-survival signaling, cell migration, dysplasia and angiogenesis. While these functions can prevent initial establishment of tumors, they can also be hijacked by aggressive cancers to enhance tumor growth and metastasis. Thus, the role of the IL-22/IL-22R1 axis in cancer is complex and context-specific. Evidence of IL-22 involvement manifests as dysregulation of IL-22 expression and signaling in patients with many common cancers including those of the gut, skin, lung and liver. Unlike other cancer-associated cytokines, IL-22 has restricted tissue specificity as its unique receptor IL-22R1 is exclusively expressed on epithelial and tissue cells, but not immune cells. This makes it an attractive target for therapy as there is potential achieve anti-tumor immunity with fewer side effects. This review summarizes current findings on functions of IL-22 in association with general mechanisms for tumorigenesis as well as specific contributions to particular cancers, and ponders how best to approach further research in the field.     

Skeletal muscle regeneration in mice is stimulated by local overexpression of V1a-vasopressin receptor

Skeletal muscle has a remarkable capacity to regenerate after mechanical or pathological injury. We show that the V1a receptor (V1aR) for vasopressin, a potent myogenic-promoting factor that stimulates differentiation and hypertrophy in vitro, is expressed in mouse skeletal muscle and modulated during regeneration after experimental injury. We used gene delivery by electroporation to overexpress the myc-tagged vasopressin V1aR in specific muscles, thus sensitizing them to circulating vasopressin. The correct localization on the surface of the fibers of the recombinant product was demonstrated by confocal immunofluorescence directed against the myc tag. V1aR overexpression dramatically enhanced regeneration. When compared with mock-transfected controls, V1aR overexpressing muscles exhibited significantly accelerated activation of satellite cells and increased expression of differentiation markers. Downstream of V1aR activation, calcineurin was strongly up-regulated and stimulated the expression of IL-4, a potent mediator of myogenic cell fusion. The central role of calcineurin in mediating V1aR-dependent myogenesis was also demonstrated by using its specific inhibitor, cyclosporine A. This study identifies skeletal muscle as a physiological target of hormones of the vasopressin family and reveals a novel in vivo role for vasopressin-dependent pathways. These findings unveil several steps, along a complex signaling pathway, that may be exploited as potential targets for the therapy of diseases characterized by altered muscle homeostasis and regeneration.     

MicroRNA-30e-5p suppresses non-small cell lung cancer tumorigenesis by regulating USP22-mediated Sirt1/JAK/STAT3 signaling

MicroRNA-30e-5p (miR-30e-5p) is a tumor suppressor that is known to be downregulated in non-small cell lung cancer (NSCLC). However, how miR-30e-5p inhibits NSCLC tumorigenesis is not known. Ubiquitin-specific peptidase 22 (USP22) is upregulated in NSCLC and promotes tumorigenesis via a Sirt1-JAK-STAT3 pathway. In this study, we investigated whether miR-30e-5p inhibits tumor growth by targeting USP22 in NSCLC. Our results reveal that miR-30e-5p expression was correlated negatively with USP22 in NSCLC tissues. Luciferase reporter assays showed that miR-30e-5p negatively regulated USP22 expression by binding to a specific sequence in the 3?UTR. MiR-30e-5p overexpression and USP22 knockdown significantly inhibited tumor growth in vivo and induced cell cycle arrest and apoptosis in NSCLC cells in vitro. The effects of miR-30e-5p inhibition were prevented by USP22 knockdown. MiR-30e-5p inhibited SIRT1 expression and increased expression of p53 and the phosphorylated form of STAT3 (pSTAT3). Furthermore, miR-30e-5p prevented USP22-mediated regulation of SIRT1, pSTAT3, and p53 expression. Taken together, these findings suggest that miR-30e-5p suppresses NSCLC tumorigenesis by downregulatingUSP22-mediated Sirt1/JAK/STAT3 signaling. Our study has identified miR-30e-5p as a potential therapeutic target for the treatment of NSCLC.     

Hyaluronan synthesis is required for IL-2-mediated T cell proliferation

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Previous studies have suggested that CD44 expressed by T cells bind exogenous HA for their proliferation. However, HA endogenously synthesized by T cells may participate in their autocrine proliferation. In this study, we examined the role of endogenous HA in T cell proliferation using the highly specific HA synthase inhibitor, 4-methylumbelliferone (4-MU). We found that 4-MU inhibited the mitogen-induced synthesis of HA by T cells. Moreover, 4-MU inhibited T cell proliferation in a dose-dependent manner when cells were cultured with different stimuli, including Con A, PMA/ionomycin, and allogeneic spleen cells. Furthermore, 4-MU inhibited mitogen-stimulated IL-2 secretion, suggesting that HA may play a role in the production of this cytokine. Addition of IL-2 to T cells treated with 4-MU and Con A reversed the block in cell proliferation, showing that impaired IL-2 production is a likely mechanism for the inhibited division of T cells. Surprisingly, an anti-CD44 Ab antagonistic for HA binding did not reduce IL-2 secretion or T cell proliferation. Importantly, 4-MU did not alter the surface expression of CD44 or the ability of CD44 to bind to HA. Thus, HA-mediated IL-2 production and T cell proliferation are CD44 independent. Our results strongly suggest that HA synthesized by T cells themselves is critical for their IL-2-mediated proliferation and have revealed a previously unrecognized role for endogenous HA in T cell biology.     

IL-22 is induced by S100/calgranulin and impairs cholesterol efflux in macrophages by downregulating ABCG1

S100A8/9 and S100A12 are emerging biomarkers for disease activity of autoimmune and cardiovascular diseases. We demonstrated previously that S100A12 accelerates atherosclerosis accompanied by large cholesterol deposits in atherosclerotic lesions of apoE-null mice. The objective of this study was to ascertain whether S100/calgranulin influences cholesterol homeostasis in macrophages. Peritoneal macrophages from transgenic mice expressing human S100A8/9 and S100A12 in myeloid cells [human bacterial artificial chromosome (hBAC)/S100] have increased lipid content and reduced ABCG1 expression and [³H]cholesterol efflux compared with WT littermates. This was associated with a 6-fold increase in plasma interleukin (IL)-22 and increased IL-22 mRNA in splenic T cells. These findings are mediated by the receptor for advanced glycation endproducts (RAGE), because hBAC/S100 mice lacking RAGE had normal IL-22 expression and normal cholesterol efflux. In vitro, recombinant IL-22 reduced ABCG1 expression and [³H]cholesterol efflux in THP-1 macrophages, while recombinant S100A12 had no effect on ABCG1 expression. In conclusion, S100/calgranulin has no direct effect on cholesterol efflux in macrophages, but rather promotes the secretion of IL-22, which then directly reduces cholesterol efflux in macrophages by decreasing the expression of ABCG1.     

Early regeneration of whole skeletal muscle grafts is unaffected by overexpression of IGF-1 in MLC/mIGF-1 transgenic mice.

Early myogenic events in regenerating whole muscle grafts were compared between transgenic MLC/mIGF-1 mice with skeletal muscle-specific overexpression of the Exon-1 Ea isoform of insulin-like growth factor-1 (mIGF-1) and control FVB mice, from day 3 to day 21 after transplantation. Immunocytochemistry with antibodies against desmin showed that skeletal muscle-specific overexpression of IGF-1 did not affect the pattern of myoblast activation or proliferation or the onset and number of myotubes formed in regenerating whole muscle grafts. Hypertrophied myotubes were observed in MLC/mIGF grafts at day 7 after transplantation, although such hypertrophy was transient, and the transgenic and control grafts had a similar appearance at later time points (days 10, 14, and 21). Immunostaining with antibodies to platelet endothelial cell adhesion molecule-1, which identifies endothelial cells, demonstrated no difference in the formation of new vascular network in grafts of transgenic and control mice. Skeletal muscle-specific overexpression of mIGF-1 does not appear to stimulate the early events associated with myogenesis during regeneration of whole muscle grafts.     

Peg3/Pw1 is involved in p53-mediated cell death pathway in brain ischemia/hypoxia.

Emerging evidence has shown that tumor suppressor p53 expression is enhanced in response to brain ischemia/hypoxia and that p53 plays a critical role in the cell death pathway in such an acute neurological insult. However the mechanism remains unclear. Recently it was reported that Peg3/Pw1, originally identified as a paternally expressed gene, plays a pivotal role in the p53-mediated cell death pathway in mouse fibroblast cell lines. In this study, we found that Peg3/Pw1 expression is enhanced in peri-ischemic neurons in rat stroke model by in situ hybridization analysis, where p53 expression was also induced by immunohistochemical analysis. Moreover, we found that p53 was co-localized with Peg3/Pw1 in brain ischemia/hypoxia by double staining analysis. In human neuroblastoma-derived SK-N-SH cells, Peg3/Pw1 mRNA expression is enhanced remarkably at 24 h post-hypoxia, when p53 protein expression was also enhanced at high levels. Subcellular localization of Peg3/Pw1 was observed in the nucleus. Adenovirus-mediated high dose p53 overexpression induced Peg3/Pw1 mRNA expression. Overexpression of Peg3/Pw1 reduced cell viability under hypoxic conditions, whereas that of the C-terminal-deleted mutant and anti-sense Peg3/Pw1 inhibited hypoxia-induced cell death. These results suggest that Peg3/Pw1 is involved in the p53-mediated cell death pathway as a downstream effector of p53 in brain ischemia/hypoxia.     

STAT3 is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production

IL-27, a member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in augmentation of Th1 differentiation and suppression of proinflammatory cytokine production. In the present study, we investigated the role of STAT3 in the IL-27-mediated immune functions. IL-27 induced phosphorylation of STAT1, -2, -3 and -5 in wild-type naive CD4+ T cells, but failed to induce that of STAT3 and STAT5 in STAT3-deficient cohorts. IL-27 induced not only proinflammatory responses including up-regulation of ICAM-1, T-box expressed in T cells, and IL-12Rbeta2 and Th1 differentiation, but also anti-inflammatory responses including suppression of proinflammatory cytokine production such as IL-2, IL-4, and IL-13 even in STAT3-deficient naive CD4+ T cells. In contrast, IL-27 augmented c-Myc and Pim-1 expression and induced cell proliferation in wild-type naive CD4+ T cells but not in STAT3-deficient cohorts. Moreover, IL-27 failed to activate STAT3, augment c-Myc and Pim-1 expression, and induce cell proliferation in pro-B BaF/3 transfectants expressing mutant gp130, in which the putative STAT3-binding four Tyr residues in the YXXQ motif of the cytoplasmic region was replaced by Phe. These results suggest that STAT3 is activated through gp130 by IL-27 and is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production. Thus, IL-27 may be a cytokine, which activates both STAT1 and STAT3 through distinct receptor subunits, WSX-1 and gp130, respectively, to mediate its individual immune functions.     

SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2

Inflammation associates with peripheral insulin resistance, which dysregulates nutrient homeostasis and leads to diabetes. Inflammation induces the expression of SOCS proteins. We show that SOCS1 or SOCS3 targeted IRS1 and IRS2, two critical signaling molecules for insulin action, for ubiquitin-mediated degradation. SOCS1 or SOCS3 bound both recombinant and endogenous IRS1 and IRS2 and promoted their ubiquitination and subsequent degradation in multiple cell types. Mutations in the conserved SOCS box of SOCS1 abrogated its interaction with the elongin BC ubiquitin-ligase complex without affecting its binding to IRS1 or IRS2. The SOCS1 mutants also failed to promote the ubiquitination and degradation of either IRS1 or IRS2. Adenoviral-mediated expression of SOCS1 in mouse liver dramatically reduced hepatic IRS1 and IRS2 protein levels and caused glucose intolerance; by contrast, expression of the SOCS1 mutants had no effect. Thus, SOCS-mediated degradation of IRS proteins, presumably via the elongin BC ubiquitin-ligase, might be a general mechanism of inflammation-induced insulin resistance, providing a target for therapy.     

Tumor necrosis factor alpha-mediated insulin resistance, but not dedifferentiation, is abrogated by MEK1/2 inhibitors in 3T3-L1 adipocytes

Tumor necrosis factor-alpha (TNFalpha) has been implicated as a contributing mediator of insulin resistance observed in pathophysiological conditions such as obesity, cancer-induced cachexia, and bacterial infections. Previous studies have demonstrated that TNFalpha confers insulin resistance by promoting phosphorylation of serine residues on insulin receptor substrate 1 (IRS-1), thereby diminishing subsequent insulin-induced tyrosine phosphorylation of IRS-1. However, little is known about which signaling molecules are involved in this process in adipocytes and about the temporal sequence of events that ultimately leads to TNFalpha-stimulated IRS-1 serine phosphorylation. In this study, we demonstrate that specific inhibitors of the MAP kinase kinase (MEK)1/2-p42/44 mitogen-activated protein (MAP) kinase pathway restore insulin signaling to normal levels despite the presence of TNFalpha. Additional experiments show that MEK1/2 activity is required for TNFalpha-induced IRS-1 serine phosphorylation, thereby suggesting a mechanism by which these inhibitors restore insulin signaling. We observe that TNFalpha requires 2.5-4 h to markedly reduce insulin-triggered tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes. Although TNFalpha activates p42/44 MAP kinase, maximal stimulation is observed within 10-30 min. To our surprise, p42/44 activity returns to basal levels well before IRS-1 serine phosphorylation and insulin resistance are observed. These activation kinetics suggest a mechanism of p42/44 action more complicated than a direct phosphorylation of IRS-1 triggered by the early spike of TNFalpha-induced p42/44 activity. Chronic TNFalpha treatment (> 72 h) causes adipocyte dedifferentiation, as evidenced by the loss of triglycerides and down-regulation of adipocyte-specific markers. We observe that this longer term TNFalpha-mediated dedifferentiation effect utilizes alternative, p42/44 MAP kinase-independent intracellular pathways. This study suggests that TNFalpha-mediated insulin resistance, but not adipocyte dedifferentiation, is mediated by the MEK1/2-p42/44 MAP kinase pathway.     

Crystal structure of the IL-22/IL-22R1 complex and its implications for the IL-22 signaling mechanism

Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9A crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2. STRUCTURED SUMMARY:     

TNFR1-mediated signaling is important to induce the improvement of liver fibrosis by bone marrow cell infusion

The importance of TNF-α signals mediated by tumor necrosis factor receptor type 1 (TNFR1) in inflammation and fibrosis induced by carbon tetrachloride (CCl₄), and in post-injury liver regeneration including a GFP/CCl₄ model developed as a liver repair model by bone marrow cell (BMC) infusion, was investigated. In mice in which TNFR1 was suppressed by antagonist administration or by knockout, liver fibrosis induced by CCl₄ was significantly decreased. In these mice, intrahepatic macrophage infiltration and TGF-β1 expression were reduced and stellate cell activity was decreased; however, expression of MMP-9 was also decreased. With GFP-positive BMC (TNFR1 wild-type, WT) infusion in these mice, fibrosis proliferation, including host endogenous intrahepatic macrophage infiltration, TGF-β1 expression and stellate cell activity, increased significantly. There was no significant increase of MMP-9 expression. In this study, TNFR1 in hosts had a promoting effect on CCl₄-induced hepatotoxicity and fibrosis, whereas BMC infusion in TNFR1 knockout mice enhanced host-derived intrahepatic inflammation and fibrosis proliferation. These findings differed from those in WT recipient mice, in which improvement in inflammation and fibrosis with BMC infusion had previously been reported. TNFR1-mediated signaling might be important to induce the improvement of liver fibrosis by bone marrow cell infusion.