Journal of Molecular Signaling最新文献

Background: Programmed necrosis is a form of caspase-independent cell death whose molecular regulation is poorly understood. While tumor necrosis factor-alpha (TNF-α) has been identified as an activator of programmed necrosis, the specific context under which this can happen is unclear. Recently we reported that TNF-α can be expressed by human tumor cells as both a membrane tethered (mTNF-α) and a soluble (sTNF-α) form. Whereas low level, tumor-derived sTNF-α acts as a tumor promoter, tumor cell expression of mTNF-α significantly delays tumor growth in mice, in large part by induction of programmed necrosis of tumor associated myeloid cells. In this study we sought to determine the molecular mechanism involved in mTNF-α oxidative stress-induced cell death by evaluating the known pathways involved in TNF receptor-induced programmed necrosis.

Methods: The source of Reactive Oxygen Species (ROS) in mTNF-α treated cells was determined by coculturing RAW 264.7 monocytic and L929 fibroblasts cells with fixed B16F10 control or mTNF-α expressing-melanoma cells in the presence of inhibitors of NADPH and mitochondria ROS. To identify the down-stream effector of TNF-a receptors (TNFR), level of phospho-RIP-1 and ceramide activity were evaluated.To determine whether mTNF-mediated cell death was dependent on a specific TNFR, cell death was measured in primary CD11b myeloid cells isolated from wild-type or TNFR-1, TNFR-2, TNFR-1 and TNFR-2 double knockout mice, cocultured with various TNF-α isoform.

Results: Tumor derived-mTNF-α increased ROS-mediated cytotoxicity, independent of caspase-3 activity. Although TNFR on target cells were required for this effect, we observed that mTNF-induced cell death could be mediated through both TNFR-1 and the death domain-lacking TNFR-2. ROS generation and cytotoxicity were inhibited by a mitochondrial respiratory chain inhibitor but not by an inhibitor of NADPH oxidase. mTNF-α mediated cytotoxicity was independent of RIP-1, a serine/threonine kinase that serves as a main adaptor protein of sTNF-α induced programmed necrosis. Instead, mTNF-α-induced ROS and cell death was prohibited by the ceramide-activated protein kinase (CAPK) inhibitor.

Conclusion: These findings demonstrate that the mTNF-α isoform is an effective inducer of programmed necrosis through a caspase independent, ceramide-related pathway. Interestingly, unlike sTNFα, mTNF-induced programmed necrosis is not dependent on the presence of TNFR1.

Background: Insulin receptors are widely distributed in the brain, where they play roles in synaptic function, memory formation, and neuroprotection. Autophosphorylation of the receptor in response to insulin stimulation is a critical step in receptor activation. In neurons, insulin stimulation leads to a rise in mitochondrial H2O2 production, which plays a role in receptor autophosphorylation. However, the kinetic characteristics of the H2O2 signal and its functional relationships with the insulin receptor during the autophosphorylation process in neurons remain unexplored to date.

Results: Experiments were carried out in culture of rat cerebellar granule neurons. Kinetic study showed that the insulin-induced H2O2 signal precedes receptor autophosphorylation and represents a single spike with a peak at 5-10 s and duration of less than 30 s. Mitochondrial complexes II and, to a lesser extent, I are involved in generation of the H2O2 signal. The mechanism by which insulin triggers the H2O2 signal involves modulation of succinate dehydrogenase activity. Insulin dose-response for receptor autophosphorylation is well described by hyperbolic function (Hill coefficient, nH, of 1.1±0.1; R2=0.99). N-acetylcysteine (NAC), a scavenger of H2O2, dose-dependently inhibited receptor autophosphorylation. The observed dose response is highly sigmoidal (Hill coefficient, nH, of 8.0±2.3; R2=0.97), signifying that insulin receptor autophosphorylation is highly ultrasensitive to the H2O2 signal. These results suggest that autophosphorylation occurred as a gradual response to increasing insulin concentrations, only if the H2O2 signal exceeded a certain threshold. Both insulin-stimulated receptor autophosphorylation and H2O2 generation were inhibited by pertussis toxin, suggesting that a pertussis toxin-sensitive G protein may link the insulin receptor to the H2O2-generating system in neurons during the autophosphorylation process.

Conclusions: In this study, we demonstrated for the first time that the receptor autophosphorylation occurs only if mitochondrial H2O2 signal exceeds a certain threshold. This finding provides novel insights into the mechanisms underlying neuronal response to insulin. The neuronal insulin receptor is activated if two conditions are met: 1) insulin binds to the receptor, and 2) the H2O2 signal surpasses a certain threshold, thus, enabling receptor autophosphorylation in all-or-nothing manner. Although the physiological rationale for this control remains to be determined, we propose that malfunction of mitochondrial H2O2 signaling may lead to the development of cerebral insulin resistance.

Background: The dependence of malignant properties of colorectal cancer (CRC) cells on IGF1R signaling has been demonstrated and several IGF1R antagonists are currently in clinical trials. Recently, we identified a novel pathway in which cAMP independent PKA activation by TGFβ signaling resulted in the destabilization of survivin/XIAP complex leading to increased cell death. In this study, we evaluated the effect of IGF1R inhibition or activation on PKA activation and its downstream cell survival signaling mechanisms.

Methods: Small molecule IGF1R kinase inhibitor OSI-906 was used to test the effect of IGF1R inhibition on PKA activation, AKAP association and its downstream cell survival signaling. In a complementary approach, ligand mediated activation of IGF1R was performed and AKAP/PKA signaling was analyzed for their downstream survival effects.

Results: We demonstrate that the inhibition of IGF1R in the IGF1R-dependent CRC subset generates cell death through a novel mechanism involving TGFβ stimulated cAMP independent PKA activity that leads to disruption of cell survival by survivin/XIAP mediated inhibition of caspase activity. Importantly, ligand mediated activation of the IGF1R in CRC cells results in the generation of cAMP dependent PKA activity that functions in cell survival by inhibiting caspase activity. Therefore, this subset of CRC demonstrates 2 opposing pathways organized by 2 different AKAPs in the cytoplasm that both utilize activation of PKA in a manner that leads to different outcomes with respect to life and death. The cAMP independent PKA activation pathway is dependent upon mitochondrial AKAP149 for its apoptotic functions. In contrast, Praja2 (Pja2), an AKAP-like E3 ligase protein was identified as a key element in controlling cAMP dependent PKA activity and pro-survival signaling. Genetic manipulation of AKAP149 and Praja2 using siRNA KD had opposing effects on PKA activity and survivin/XIAP regulation.

Conclusions: We had identified 2 cytoplasmic pathways dependent upon the same enzymatic activity with opposite effects on cell fate in terms of life and death. Understanding the specific mechanistic functions of IGF1R with respect to determining the PKA survival functions would have potential for impact upon the development of new therapeutic strategies by exploiting the IGF1R/cAMP-PKA survival signaling in cancer.

Background: Protrusions of cancer cells conferrers a vital function for cell migration and metastasis. Protein and RNA localization mechanisms have been extensively examined and shown to play pivotal roles for the functional presence of specific protein components in cancer cell protrusions.

Methods: To describe genome wide RNA localized in protrusions of the metastatic human breast cancer cell line MDA-MB-231 we used Boyden chamber based methodology followed by direct mRNA sequencing.

Results: In the hereby identified group of protrusion localized mRNA some previously were described to be localized exemplified by mRNA for Ras-Related protein 13 (RAB13) and p0071 (Plakophilin-4/PKP4). For other transcripts, exemplified by mRNA for SH3PXD2A/TKS5 and PPFIA1/Liprin-α1, only the corresponding proteins previously were described to have protrusion localization. Finally, a cohort of MDA-MB-231 protrusion localized transcripts represents novel candidates to mediate cancer cell subcellular region specific functions through mRNA direction to protrusions. We included a further characterization of p0071, an armadillo repeat protein of adherence junctions and desmosomes, in MDA-MB-231 and non-metastatic MCF7 cells including analysis of novel identified alternative spliced p0071 mRNA isoforms. The results showed isoform and cell type specific p0071 mRNA localization.

Conclusions: Altogether, the presented data represents a genome wide and gene specific descriptive and functional analyses of RNA localization in protrusions of MDA-MB-231 metastatic cancer cells.

Background: Tks5/FISH is a scaffold protein comprising of five SH3 domains and one PX domain. Tks5 is a substrate of the tyrosine kinase Src and is required for the organization of podosomes/invadopodia implicated in invasion of tumor cells. Recent data have suggested that a close homologue of Tks5, Tks4, is implicated in the EGF signaling.

Results: Here, we report that Tks5 is a component of the EGF signaling pathway. In EGF-treated cells, Tks5 is tyrosine phosphorylated within minutes and the level of phosphorylation is sustained for at least 2 hours. Using specific kinase inhibitors, we demonstrate that tyrosine phosphorylation of Tks5 is catalyzed by Src tyrosine kinase. We show that treatment of cells with EGF results in plasma membrane translocation of Tks5. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutation of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks5.

Conclusions: Our results identify Tks5 as a novel component of the EGF signaling pathway.

The tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of intracellular proteins were originally identified as signaling adaptors that bind directly to the cytoplasmic regions of receptors of the TNF-R superfamily. The past decade has witnessed rapid expansion of receptor families identified to employ TRAFs for signaling. These include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), T cell receptor, IL-1 receptor family, IL-17 receptors, IFN receptors and TGFβ receptors. In addition to their role as adaptor proteins, most TRAFs also act as E3 ubiquitin ligases to activate downstream signaling events. TRAF-dependent signaling pathways typically lead to the activation of nuclear factor-κBs (NF-κBs), mitogen-activated protein kinases (MAPKs), or interferon-regulatory factors (IRFs). Compelling evidence obtained from germ-line and cell-specific TRAF-deficient mice demonstrates that each TRAF plays indispensable and non-redundant physiological roles, regulating innate and adaptive immunity, embryonic development, tissue homeostasis, stress response, and bone metabolism. Notably, mounting evidence implicates TRAFs in the pathogenesis of human diseases such as cancers and autoimmune diseases, which has sparked new appreciation and interest in TRAF research. This review presents an overview of the current knowledge of TRAFs, with an emphasis on recent findings concerning TRAF molecules in signaling and in human diseases.

Background: Ovarian cancer G protein coupled receptor 1 (OGR1) mediates inhibitory effects on cell migration in human prostate and ovarian cancer cells. However, the mechanisms and signaling pathways that mediate these inhibitory effects are essentially unknown.

Methods: MCF7 cell line was chosen as a model system to study the mechanisms by which OGR1 regulates cell migration, since it expresses very low levels of endogenous OGR1. Cell migratory activities were assessed using both wound healing and transwell migration assays. The signaling pathways involved were studied using pharmacological inhibitors and genetic forms of the relevant genes, as well as small G protein pull-down activity assays. The expression levels of various signaling molecules were analyzed by Western blot and quantitative PCR analysis.

Results: Over-expression of OGR1 in MCF7 cells substantially enhanced activation of Rho and inhibition of Rac1, resulting in inhibition of cell migration. In addition, expression of the Gα12/13 specific regulator of G protein signaling (RGS) domain of p115RhoGEF, but not treatment with pertussis toxin (PTX, a Gαi specific inhibitor), could abrogate OGR1-dependent Rho activation, Rac1 inactivation, and inhibition of migration in MCF7 cells. The bioactive lipids tested had no effect on OGR1 function in cell migration.

Conclusion: Our data suggest, for the first time, that OGR1 inhibits cell migration through a Gα12/13 -Rho-Rac1 signaling pathway in MCF7 cells. This pathway was not significantly affected by bioactive lipids and all the assays were conducted at constant pH, suggesting a constitutive activity of OGR1. This is the first clear delineation of an OGR1-mediated cell signaling pathway involved in migration.

The Nuclear Factor of Activated T Cells-5 (NFAT5), also known as OREBP or TonEBP, is a member of the nuclear factors of the activated T cells family of transcription factors. It is also the only known tonicity-regulated transcription factor in mammals. NFAT5 was initially known for its role in the hypertonic kidney inner medulla for orchestrating a genetic program to restore the cellular homeostasis. Emerging evidence, however, suggests that NFAT5 might play a more diverse functional role, including a pivotal role in blood pressure regulation and the development of autoimmune diseases. Despite the growing significance of NFAT5 in physiology and diseases, our understanding of how its activity is regulated remains very limited. Furthermore, how changes in tonicities are converted into functional outputs via NFAT5 remains elusive. Therefore, this review aims to summarize our current knowledge on the functional roles of NFAT5 in osmotic stress adaptation and the signaling pathways that regulate its activity.

Background: The phosphoinositide 3-kinase (PI3K)/Akt pathway is involved in neuroblastoma development where Akt/PKB activation is associated with poor prognosis. PI3K activity subsequently activates Akt/PKB, and as mutations of PI3K are rare in neuroblastoma and high levels of PI3K subunit p110delta is associated with favorable disease with low p-Akt/PKB, the levels of other PI3K subunits could be important for Akt activation.

Methods: Protein levels of Type IA PI3K catalytic and regulatory subunits were investigated together with levels of phosphorylated Akt/PKB and the PI3K negative regulator PTEN in primary neuroblastoma tumors. Relation between clinical markers and protein levels were evaluated through t-tests.

Results: We found high levels of p-Akt/PKB correlating to aggressive disease and p-Akt/PKB (T308) showed inverse correlation to PTEN levels. The regulatory isomers p55alpha/p50alpha showed higher levels in favorable neuroblastoma as compared with aggressive neuroblastoma. The PI3K-subunit p110alpha was found mainly in advanced tumors while p110delta showed higher levels in favorable neuroblastoma.

Conclusions: Activation of the PI3K/Akt pathway is seen in neuroblastoma tumors, however the contribution of the different PI3K isoforms is unknown. Here we show that p110alpha is preferentially expressed in aggressive neuroblastomas, with high p-Akt/PKB and p110delta is mainly detected in favorable neuroblastomas, with low p-Akt/PKB. This is an important finding as PI3K-specific inhibitors are suggested for enrollment in treatment of neuroblastoma patients.

Background: Heterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the α-subunits Gα12 and Gα13, stimulate the monomeric G protein RhoA through interaction with a distinct subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs). The structural features that mediate interaction between Gα13 and RhoGEFs have been examined in crystallographic studies of the purified complex, whereas a Gα12:RhoGEF complex has not been reported. Several signaling responses and effector interactions appear unique to Gα12 or Gα13, despite their similarity in amino acid sequence.

Methods: To comprehensively examine Gα12 for regions involved in RhoGEF interaction, we screened a panel of Gα12 cassette substitution mutants for binding to leukemia-associated RhoGEF (LARG) and for activation of serum response element mediated transcription.

Results: We identified several cassette substitutions that disrupt Gα12 binding to LARG and the related p115RhoGEF. These Gα12 mutants also were impaired in activating serum response element mediated signaling, a Rho-dependent response. Most of these mutants matched corresponding regions of Gα13 reported to contact p115RhoGEF, but unexpectedly, several RhoGEF-uncoupling mutations were found within the N- and C-terminal regions of Gα12. Trypsin protection assays revealed several mutants in these regions as retaining conformational activation. In addition, charge substitutions near the Gα12 N-terminus selectively disrupted binding to LARG but not p115RhoGEF.

Conclusions: Several structural aspects of the Gα12:RhoGEF interface differ from the reported Gα13:RhoGEF complex, particularly determinants within the C-terminal α5 helix and structurally uncharacterized N-terminus of Gα12. Furthermore, key residues at the Gα12 N-terminus may confer selectivity for LARG as a downstream effector.