Determinants of phosphatidylinositol-4-phosphate 5-kinase type Iγ90 uropod location in T-lymphocytes and its role in uropod formation

We have previously identified phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90 as a T cell uropod component. However, the molecular determinants and functional consequences of its localization remain unknown. In this report, we seek to better understand the mechanisms involved in PIPKIγ90 uropod targeting and the role that PIPKIγ90 plays in T cell uropod formation. During T cell activation, PIPKIγ90 cocaps with the membrane microdomain-associated proteins flotillin-1 and -2 and accumulates in the uropod. We report that the C-terminal 26 amino acid extension of PIPKIγ90 is required for its localization to the uropod. We further use T cells from PIPKIγ90(-/-) mice and human T cells expressing a kinase-dead PIPKIγ90 mutant to examine the role of PIPKIγ90 in a T cell uropod formation. We find that PIPKIγ90 deficient T cells have elongated uropods on ICAM-1. Moreover, in human T cells overexpression of PIPKIγ87, a naturally occurring isoform lacking the last 26 amino acids, suppresses uropod formation and impairs capping of uropod proteins such as flotillins. Transfection of human T cells with a dominant-negative mutant of flotillin-2 in turn attenuates capping of PIPKIγ90. Our data contribute to the understanding of the molecular mechanisms that regulate T cell uropod formation.

Epidermal growth factor receptor, phosphatidylinositol-3-kinase catalytic subunit/PTEN, and KRAS/NRAS/BRAF in primary resected esophageal adenocarcinomas: loss of PTEN is associated with worse clinical outcome

Alterations of the epidermal growth factor receptor (EGFR) can be observed in a significant subset of esophageal adenocarcinomas (EACs), and targeted therapy against EGFR may become an interesting approach for the treatment of these tumors. Mutations of KRAS, NRAS, BRAF, and phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) and deregulation of PTEN expression influence the responsiveness against anti-EGFR therapy in colorectal carcinomas. We investigated the prevalence of these events in a collection of 117 primary resected EACs, correlated the findings with EGFR expression and amplification, and determined their clinicopathologic impact. KRAS mutations were detected in 4 (3%) of 117 tumors (3× G12D and 1 G12V mutation). One tumor had a PIK3CA E545K mutation. Neither NRAS nor BRAF mutations were detected. Sixteen (14%) of 117 cases were negative for PTEN expression, determined by immunohistochemistry. Loss of PTEN was observed predominantly in advanced tumor stages (P = .004). There was no association between PTEN and EGFR status. Loss of PTEN was associated with shorter overall and disease-free survival (P < .001 each) and also an independent prognostic factor in multivariate analysis (P = .015). EGFR status had no prognostic impact in this case collection. In summary, loss of PTEN can be detected in a significant subset of EAC and is associated with an aggressive phenotype. Therefore, PTEN may be useful as a prognostic biomarker. In contrast, mutations of RAS/RAF/PIK3CA appear only very rarely, if at all, in EAC. A possible predictive role of PTEN in anti-EGFR treatment warrants further investigations, whereas determination of RAS/RAF/PIK3CA mutations may only have a minor impact in this context.

TGF-βRI kinase activity mediates Emdogain-stimulated in vitro osteoclastogenesis.

OBJECTIVES Emdogain, containing an extract of fetal porcine enamel matrix proteins, is a potent stimulator of in vitro osteoclastogenesis. The underlying molecular mechanisms are, however, unclear. MATERIAL AND METHODS Here, we have addressed the role of transforming growth factor-beta receptor type 1 (TGF-βRI) kinase activity on osteoclastogenesis in murine bone marrow cultures. RESULTS Inhibition of TGF-βRI kinase activity with SB431542 abolished the effect of Emdogain on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand or tumor necrosis factor-alpha. SB431542 also suppressed the Emdogain-mediated increase of OSCAR, a co-stimulatory protein, and dendritic cell-specific transmembrane protein and Atp6v0d2, the latter two being involved in cell fusion. Similar to transforming growth factor-beta1 (TGF-β), Emdogain could not compensate for the inhibition of IL-4 and IFNγ on osteoclast formation. When using the murine macrophage cell line RAW246.7, SB431542 and the smad-3 inhibitor SIS3 blocked Emdogain-stimulated expression of the transcription factor NFATc1. CONCLUSIONS Taken together, the data suggest that TGF-βRI kinase activity is necessary to mediate in vitro effects of Emdogain on osteoclastogenesis. CLINICAL RELEVANCE Based on these in vitro data, we can speculate that at least part of the clinical effects of Emdogain on osteoclastogenesis is mediated via TGF-β signaling.

The Rice Transcription Factor WRKY53 Suppresses Herbivore-Induced Defenses by Acting as a Negative Feedback Modulator of Mitogen-Activated Protein Kinase Activity

The mechanisms by which herbivore-attacked plants activate their defenses are well studied. By contrast, little is known about the regulatory mechanisms that allow them to control their defensive investment and avoid a defensive overshoot. We characterized a rice (Oryza sativa) WRKY gene, OsWRKY53, whose expression is rapidly induced upon wounding and induced in a delayed fashion upon attack by the striped stem borer (SSB) Chilo suppressalis. The transcript levels of OsWRKY53 are independent of endogenous jasmonic acid but positively regulated by the mitogen-activated protein kinases OsMPK3/OsMPK6. OsWRKY53 physically interacts with OsMPK3/OsMPK6 and suppresses their activity in vitro. By consequence, it modulates the expression of defensive, MPK-regulated WRKYs and thereby reduces jasmonic acid, jasmonoyl-isoleucine, and ethylene induction. This phytohormonal reconfiguration is associated with a reduction in trypsin protease inhibitor activity and improved SSB performance. OsWRKY53 is also shown to be a negative regulator of plant growth. Taken together, these results show that OsWRKY53 functions as a negative feedback modulator of MPK3/MPK6 and thereby acts as an early suppressor of induced defenses. OsWRKY53 therefore enables rice plants to control the magnitude of their defensive investment during early signaling.

Phosphatidylinositol 3-kinase isoforms as novel drug targets

Phosphatidylinositol 3-kinases (PI3Ks) are key molecules in the signal transduction pathways initiated by the binding of extracellular signals to their cell surface receptors. The PI3K family of enzymes comprises eight catalytic isoforms subdivided into three classes and control a variety of cellular processes including proliferation, growth, apoptosis, migration and metabolism. Deregulation of the PI3K pathway has been extensively investigated in connection to cancer, but is also involved in other commonly occurring diseases such as chronic inflammation, autoimmunity, allergy, atherosclerosis, cardiovascular and metabolic diseases. The fact that the PI3K pathway is deregulated in a large number of human diseases, and its importance for different cellular responses, makes it an attractive drug target. Pharmacological PI3K inhibitors have played a very important role in studying cellular responses involving these enzymes. Currently, a wide range of selective PI3K inhibitors have been tested in preclinical studies and some have entered clinical trials in oncology. However, due to the complexity of PI3K signaling pathways, developing an effective anti-cancer therapy may be difficult. The biggest challenge in curing cancer patients with various signaling pathway abnormalities is to target multiple components of different signal transduction pathways with mechanism-based combinatorial treatments. In this article we will give an overview of the complex role of PI3K isoforms in human diseases and discuss their potential as drug targets. In addition, we will describe the drugs currently used in clinical trials, as well as promising emerging candidates.

Proviral integration site for Moloney murine leukemia virus 1, but not phosphatidylinositol-3 kinase, is essential in the antiapoptotic signaling cascade initiated by IL-5 in eosinophils

BACKGROUND: Eosinophil differentiation, activation, and survival are largely regulated by IL-5. IL-5-mediated transmembrane signal transduction involves both Lyn-mitogen-activated protein kinases and Janus kinase 2-signal transducer and activator of transcription pathways. OBJECTIVE: We sought to determine whether additional signaling molecules/pathways are critically involved in IL-5-mediated eosinophil survival. METHODS: Eosinophil survival and apoptosis were measured in the presence and absence of IL-5 and defined pharmacologic inhibitors in vitro. The specific role of the serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim) 1 was tested by using HIV-transactivator of transcription fusion proteins containing wild-type Pim-1 or a dominant-negative form of Pim-1. The expression of Pim-1 in eosinophils was analyzed by means of immunoblotting and immunofluorescence. RESULTS: Although pharmacologic inhibition of phosphatidylinositol-3 kinase (PI3K) by LY294002, wortmannin, or the selective PI3K p110delta isoform inhibitor IC87114 was successful in each case, only LY294002 blocked increased IL-5-mediated eosinophil survival. This suggested that LY294002 inhibited another kinase that is critically involved in this process in addition to PI3K. Indeed, Pim-1 was rapidly and strongly expressed in eosinophils after IL-5 stimulation in vitro and readily detected in eosinophils under inflammatory conditions in vivo. Moreover, by using specific protein transfer, we identified Pim-1 as a critical element in IL-5-mediated antiapoptotic signaling in eosinophils. CONCLUSIONS: Pim-1, but not PI3K, plays a major role in IL-5-mediated antiapoptotic signaling in eosinophils.

Enamel matrix derivative inhibits adipocyte differentiation of 3T3-L1 cells via activation of TGF-βRI kinase activity

Enamel matrix derivative (EMD), an extract of fetal porcine enamel, and TGF-β can both suppress adipogenic differentiation. However, there have been no studies that functionally link the role of EMD and TGF-β in vitro. Herein, we examined whether TGF-β signaling contributes to EMD-induced suppression of adipogenic differentiation. Adipogenesis was studied with 3T3-L1 preadipocytes in the presence of SB431542, an inhibitor of TGF-βRI kinase activity. SB431542 reversed the inhibitory effect of EMD on adipogenic differentiation, based on Oil Red O staining and mRNA expression of lipid regulated genes. SB431542 also reduced EMD-stimulated expression of connective tissue growth factor (CTGF), an autocrine inhibitor of adipogenic differentiation. Moreover, short interfering (si)RNAs for CTGF partially reversed the EMD-induced suppression of lipid regulated genes. We conclude that the TGF-βRI - CTGF axis is involved in the anti-adipogenic effects of EMD in vitro.

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca(2+) release and store-operated Ca(2+) entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1microM) and the Src inhibitor PP2 (10microM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca(2+) transients were reduced by imatinib and/or PP2. Ca(2+) transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca(2+) transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCalpha catalytic activity and PKCalpha co-immunoprecipitated with Bcr/Abl. Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca(2+) influx was reduced by complexing extracellular Ca(2+) with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca(2+) transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.

Effects of troglitazone on cellular differentiation, insulin signaling, and glucose metabolism in cultured human skeletal muscle cells

To determine the immediate effect of thiazolidinediones on human skeletal muscle, differentiated human myotubes were acutely (1 day) and myoblasts chronically (during the differentiation process) treated with troglitazone (TGZ). Chronic TGZ treatment resulted in loss of the typical multinucleated phenotype. The increase of muscle markers typically observed during differentiation was suppressed, while adipocyte markers increased markedly. Chronic TGZ treatment increased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and membranous protein kinase B/Akt (PKB/Akt) Ser-473 phosphorylation more than 4-fold. Phosphorylation of p42/44 mitogen-activated protein kinase (42/44 MAPK/ERK) was unaltered. Basal glucose uptake as well as both basal and insulin-stimulated glycogen synthesis increased approximately 1.6- and approximately 2.5-fold after chronic TGZ treatment, respectively. A 2-fold stimulation of PI 3-kinase but no other significant TGZ effect was found after acute TGZ treatment. In conclusion, chronic TGZ treatment inhibited myogenic differentiation of that human muscle while inducing adipocyte-specific gene expression. The effects of chronic TGZ treatment on basal glucose transport may in part be secondary to this transdifferentiation. The enhancing effect on PI 3-kinase and PKB/Akt involved in both differentiation and glycogen synthesis appears to be pivotal in the cellular action of TGZ.

Ramipril increases the protein level of skeletal muscle IRS-1 and alters protein tyrosine phosphatase activity in spontaneously hypertensive rats

To investigate mechanisms by which angiotensin converting enzyme (ACE)-inhibition increases insulin sensitivity, spontaneously hypertensive (SH) rats were treated with or without ramipril (1 mg/kg per day) for 12 weeks. Insulin binding and protein levels of insulin receptor substrate-1 (IRS-1), p85-subunit of phosphatidylinositol 3'-kinase (p85) and Src homology 2 domain-containing phosphatase-2 (SHP2) were then determined in hindlimb muscle and liver. Additionally, protein tyrosine phosphatase (PTPase) activities towards immobilized phosphorylated insulin receptor or phosphorylated IRS-1 of membrane (MF) and cytosolic fractions (CF) of these tissues were measured. Ramipril treatment increased IRS-1-protein content in muscle by 31+/-9% (P<0.05). No effects were observed on IRS-1 content in liver or on insulin binding or protein expression of p85 or SHP2 in both tissues. Ramipril treatment also increased dephosphorylation of insulin receptor by muscle CF (22.0+/-1.0%/60 min compared to 16.8+/-1.5%/60 min; P<0.05), and of IRS-1 by liver MF (37.2+/-1.7%/7.5 min compared to 33.8+/-1.7%/7.5 min; P<0.05) and CF (36.8+/-1.0%/7.5 min compared to 33.2+/-1.0%/7.5 min; P<0.05). We conclude that the observed effects of ACE-inhibition by ramipril on the protein expression of IRS-1 and on PTPase activity might contribute to its effect on insulin sensitivity.

Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms beta2 and theta

AIMS/HYPOTHESIS: Inhibition of the signalling function of the human insulin receptor (HIR) is one of the principle mechanisms which induce cellular insulin resistance. It is speculated that serine residues in the insulin receptor beta-subunit are involved in receptor inhibition either as inhibitory phosphorylation sites or as part of receptor domains which bind inhibitory proteins or tyrosine phosphatases. As reported earlier we prepared 16 serine to alanine point mutations of the HIR and found that serine to alanine mutants HIR-994 and HIR-1023/25 showed increased tyrosine autophosphorylation when expressed in human embryonic kidney (HEK) 293 cells. In this study we examined whether these mutant receptors have a different susceptibility to inhibition by serine kinases or an altered tyrosine kinase activity. METHODS: Tyrosine kinase assay and transfection studies. RESULTS: In an in vitro kinase assay using IRS-1 as a substrate we could detect a higher intrinsic tyrosine kinase activity of both receptor constructs. Additionally, a higher capacity to phosphorylate the adapter protein Shc in intact cells was seen. To test the inhibition by serine kinases, the receptor constructs were expressed in HEK 293 cells together with IRS-1 and protein kinase C isoforms beta2 and theta. Phorbol ester stimulation of these cells reduced wild-type receptor autophosphorylation to 58 % or 55 % of the insulin simulated state, respectively. This inhibitory effect was not observed with HIR-994 and HIR-1023/25, although all other tested HIR mutants showed similar inhibition induced by protein kinase C. CONCLUSION/INTERPRETATION: The data suggest that the HIR-domain which contains the serine residues 994 and 1023/25 is important for the inhibitory effect of protein kinase C isoforms beta2 and theta on insulin receptor autophosphorylation.

Antiseptic solutions modulate the paracrine-like activity of bone chips: differential impact of chlorhexidine and sodium hypochlorite

AIM: Chemical decontamination increases the availability of bone grafts; however, it is unclear whether antiseptic processing changes the biological activity of bone. MATERIALS AND METHODS: Bone chips were incubated with 4 different antiseptic solutions including (1) povidone-iodine (0.5%), (2) chlorhexidine diguluconate (0.2%), (3) hydrogen peroxide (1%) and (4) sodium hypochlorite (0.25%). After 10 minutes of incubation, changes in the capacity of the bone-conditioned medium to modulate gene expression of gingival fibroblasts was investigated. RESULTS: Conditioned medium obtained from freshly prepared bone chips increased the expression of TGF-β target genes interleukin 11 (IL11), proteoglycan4 (PRG4), NADPH oxidase 4 (NOX4), and decreased the expression of adrenomedullin (ADM), and pentraxin 3 (PTX3) in gingival fibroblasts. Incubation of bone chips with 0.2% chlorhexidine, followed by vigorously washing resulted in a bone-conditioned medium with even higher expression of IL11, PRG4, and NOX4. These findings were also found with a decrease in cell viability and an activation of apoptosis signaling. Chlorhexidine alone, at low concentrations, increased IL11, PRG4 and NOX4 expression, independent of the TGF-β receptor I kinase activity. In contrast, 0.25% sodium hypochlorite almost entirely abolished the activity of bone-conditioned medium, while the other two antiseptic solutions, 1% hydrogen peroxide and 0.5% povidone-iodine, had relatively no impact, respectively. CONCLUSION: These in vitro findings demonstrate that incubation of bone chips with chlorhexidine differentially affects the activity of the respective bone-conditioned medium compared to the other antiseptic solutions. The data further suggest that the main effects are caused by chlorhexidine remaining in the bone-conditioned medium after repeated washing of the bone chips. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. KEYWORDS: Autografts; TGF-β; antiseptic solution; bone; bone conditioned medium; bone supernatant; chlorhexidine; hydrogen peroxide; povidone-iodine; sodium hypochlorite

Targeting the insulin-like growth factor-1 receptor in human cancer

The insulin-like growth factor (IGF) signaling system plays a crucial role in human cancer and the IGF-1 receptor (IGF-1R) is an attractive drug target against which a variety of novel anti-tumor agents are being developed. Deregulation of the IGF signaling pathway frequently occurs in human cancer and involves the establishment of autocrine loops comprising IGF-1 or IGF-2 and/or IGF-1R over-expression. Epidemiologic studies have documented a link between elevated IGF levels and the development of solid tumors, such as breast, colon, and prostate cancer. Anti-cancer strategies targeting the IGF signaling system involve two main approaches, namely neutralizing antibodies and small molecule inhibitors of the IGF-1R kinase activity. There are numerous reports describing anti-tumor activity of these agents in pre-clinical models of major human cancers. In addition, multiple clinical trials have started to evaluate the safety and efficacy of selected IGF-1R inhibitors, in combination with standard chemotherapeutic regimens or other targeted agents in cancer patients. In this mini review, I will discuss the role of the IGF signaling system in human cancer and the main strategies which have been so far evaluated to target the IGF-1R.

Patterns of functional enzyme activity in fungus farming ambrosia beetles

Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi) indicates that only larvae (pre-) digest plant cell wall structures. This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.