Notch1 Signaling Sensitizes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Human Hepatocellular Carcinoma Cells by Inhibiting Akt/Hdm2-mediated p53 Degradation and Up-regulating p53-dependent DR5 Expression

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC.Notch signaling determines cell fate and affects cell proliferation, differentiation, and apoptosis during cell development (1). As a highly conserved family, Notch coordinates a signaling cascade present in all animal species studied to date (2). Mammals have four Notch receptors that bind five different ligands, among which Notch1 signaling functions in many physiological and pathophysiological processes of numerous cell types, and its dysfunction results in a variety of developmental defects, including embryonic lethality and adult disorders. For example, the Notch1/Jagged1 signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting hepatocyte growth (3, 4). Inducible inactivation of Notch1 has been shown to cause nodular regenerative hyperplasia in mouse liver (5). These studies suggest that Notch1 signaling may be involved in the liver functions and the pathogenesis of liver diseases. Our previous study demonstrated that Notch1 signaling could suppress the growth of human hepatocellular carcinoma (HCC)⁴ cells by arresting the cell cycle and inducing apoptosis (6). However, the underlying molecular mechanisms remain to be fully understood.p53, an important tumor suppressor gene, is involved in cell cycle arrest and cellular apoptosis. Its activity is mostly regulated by complex networks of post-translational modifications, including phosphorylation, ubiquitination, and proteasome degradation. One protein that is essential for determining p53 stability is Mdm2 (mouse double minute protein 2) (7). Mdm2, a nuclear phosphoprotein and an E3 ubiquitin ligase, binds to p53 and ubiquitinates p53, leading to proteosome degradation of p53 (8). Another important mechanism of p53 stability is related to its phosphorylation status, which is Mdm2-dependent or Mdm2-independent (9). As to the regulation of p53 by Notch1, there are controversial reports that Notch1 activation increased p53 expression in neural progenitor cells (10); however, suppression of p53 by Notch signaling was also well established in lymphomagenesis (11). We also reported that Notch1 signaling significantly up-regulated p53 expression in SMMC7721 HCC cells (6); however, the molecular mechanisms remained unclear and needed to be further characterized.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of a superfamily of cell death-inducing ligands, induces apoptosis in a broad range of transformed cells and tumor cells but has little or no effect on normal cells (12). Therefore, TRAIL has been regarded as a potential drug for cancer therapy (12, 13). However, several kinds of cancer, including HCC, are not sensitive to soluble TRAIL treatment (14). HCC accounts for 80–90% of liver cancers and is one of the most prevalent carcinomas throughout the world, especially in Africa and Asia. Thus, it is worthwhile to find a new strategy to overcome the resistance of HCC cells to TRAIL-induced apoptosis.Considering that Notch1 signaling up-regulates p53 and induces apoptosis of HCC cells and that there are no reports to date that address the relationship between Notch1 signaling and TRAIL-induced apoptosis, in this study, we investigated whether and how Notch1 signaling could sensitize HCC cells to TRAIL-induced apoptosis. We demonstrate that Notch1 signaling up-regulates p53 expression by inhibiting proteasome degradation via, at least in part, suppressing the phosphatidylinositol 3-kinase/Akt/Hdm2 pathway. In addition, we here report that Notch1 signaling enhances DR5 (death receptor 5) expression in a p53-dependent manner, and DR5 contributes, at least in part, to the enhancement of TRAIL-induced apoptosis by Notch1 signaling. Accordingly, Notch1 signaling sensitizes HCC cells to TRAIL-induced apoptosis.     

Biased agonism of the calcium-sensing receptor

After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby "biasing" the signaling, it has become obvious that the original model of GPCRs existing in either an "on" or "off" conformation is too simple. The current explanation for this biased agonism is that GPCRs can adopt multiple active conformations stabilized by different molecules, and that each conformation affects intracellular signaling in a different way. In the present study we sought to investigate biased agonism of the calcium-sensing receptor (CaSR), by looking at 12 well-known orthosteric CaSR agonists in 3 different CaSR signaling pathways: G(q/11) protein, G(i/o) protein, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Here we show that apart from G(q/11) and G(i/o) signaling, ERK1/2 is activated through recruitment of β-arrestins. Next, by measuring activity of all three signaling pathways we found that barium, spermine, neomycin, and tobramycin act as biased agonist in terms of efficacy and/or potency. Finally, polyamines and aminoglycosides in general were biased in their potencies toward ERK1/2 signaling. In conclusion, the results of this study indicate that several active conformations of CaSR, stabilized by different molecules, exist, which affect intracellular signaling distinctly.     

Beclin 1 regulates neuronal transforming growth factor-β signaling by mediating recycling of the type I receptor ALK5

Background

Beclin 1 is a key regulator of multiple trafficking pathways, including autophagy and receptor recycling in yeast and microglia. Decreased beclin 1 levels in the CNS result in neurodegeneration, an effect attributed to impaired autophagy. However, neurons also rely heavily on trophic factors, and signaling through these pathways requires the proper trafficking of trophic factor receptors.

Results

We discovered that beclin 1 regulates signaling through the neuroprotective TGF-β pathway. Beclin 1 is required for recycling of the type I TGF-β receptor ALK5. We show that beclin 1 recruits the retromer to ALK5 and facilitates its localization to Rab11⁺ endosomes. Decreased levels of beclin 1, or its binding partners VPS34 and UVRAG, impair TGF-β signaling.

Conclusions

These findings identify beclin 1 as a positive regulator of a trophic signaling pathway via receptor recycling, and suggest that neuronal death induced by decreased beclin 1 levels may also be due to impaired trophic factor signaling.

     

Molecular basis of Wnt activation via the DIX domain protein Ccd1

The Wnt signaling plays pivotal roles in embryogenesis and cancer, and the three DIX domain-containing proteins, Dvl, Axin, and Ccd1, play distinct roles in the initiation and regulation of canonical Wnt signaling. Overexpressed Dvl has a tendency to form large polymers in a cytoplasmic punctate pattern, whereas the biologically active Dvl in fact forms low molecular weight oligomers. The molecular basis for how the polymeric sizes of Dvl proteins are controlled upon Wnt signaling remains unclear. Here we show that Ccd1 up-regulates canonical Wnt signaling via acting synergistically with Dvl. We determined the crystal structures of wild type Ccd1-DIX and mutant Dvl1-DIX(Y17D), which pack into "head-to-tail" helical filaments. Structural analyses reveal two sites crucial for intra-filament homo- and hetero-interaction and a third site for inter-filament homo-assembly. Systematic mutagenesis studies identified critical residues from all three sites required for Dvl homo-oligomerization, puncta formation, and stimulation of Wnt signaling. Remarkably, Ccd1 forms a hetero-complex with Dvl through the "head" of Dvl-DIX and the "tail" of Ccd1-DIX, depolymerizes Dvl homo-assembly, and thereby controls the size of Dvl polymer. These data together suggest a molecular mechanism for Ccd1-mediated Wnt activation in that Ccd1 converts latent polymeric Dvl to a biologically active oligomer(s).     

Predictive model identifies strategies to enhance TSP1-mediated apoptosis signaling

Background

Thrombospondin-1 (TSP1) is a matricellular protein that functions to inhibit angiogenesis. An important pathway that contributes to this inhibitory effect is triggered by TSP1 binding to the CD36 receptor, inducing endothelial cell apoptosis. However, therapies that mimic this function have not demonstrated clear clinical efficacy. This study explores strategies to enhance TSP1-induced apoptosis in endothelial cells. In particular, we focus on establishing a computational model to describe the signaling pathway, and using this model to investigate the effects of several approaches to perturb the TSP1-CD36 signaling network.

Methods

We constructed a molecularly-detailed mathematical model of TSP1-mediated intracellular signaling via the CD36 receptor based on literature evidence. We employed systems biology tools to train and validate the model and further expanded the model by accounting for the heterogeneity within the cell population. The initial concentrations of signaling species or kinetic rates were altered to simulate the effects of perturbations to the signaling network.

Results

Model simulations predict the population-based response to strategies to enhance TSP1-mediated apoptosis, such as downregulating the apoptosis inhibitor XIAP and inhibiting phosphatase activity. The model also postulates a new mechanism of low dosage doxorubicin treatment in combination with TSP1 stimulation. Using computational analysis, we predict which cells will undergo apoptosis, based on the initial intracellular concentrations of particular signaling species.

Conclusions

This new mathematical model recapitulates the intracellular dynamics of the TSP1-induced apoptosis signaling pathway. Overall, the modeling framework predicts molecular strategies that increase TSP1-mediated apoptosis, which is useful in many disease settings.

     

Leucine Facilitates Insulin Signaling through a Gαi Protein-dependent Signaling Pathway in Hepatocytes

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt⁴⁷³ and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002), Gα_i protein (pertussis toxin or siRNA against Gα_i1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gα_i1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gα_i protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly.     

Arabidopsis Phytochrome B Promotes SPA1 Nuclear Accumulation to Repress Photomorphogenesis under Far-Red Light

Phytochrome A (phyA) is the primary photoreceptor mediating deetiolation under far-red (FR) light, whereas phyB predominantly regulates light responses in red light. SUPPRESSOR OF PHYA-105 (SPA1) forms an E3 ubiquitin ligase complex with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), which is responsible for the degradation of various photomorphogenesis-promoting factors, resulting in desensitization to light signaling. However, the role of phyB in FR light signaling and the regulatory pathway from light-activated phytochromes to the COP1-SPA1 complex are largely unknown. Here, we confirm that PHYB overexpression causes an etiolation response with reduced ELONGATED HYPOCOTYL5 (HY5) accumulation under FR light. Notably, phyB exerts its nuclear activities and promotes seedling etiolation in both the presence and absence of phyA in response to FR light. PhyB acts upstream of SPA1 and is functionally dependent on it in FR light signaling. PhyB interacts and forms a protein complex with SPA1, enhancing its nuclear accumulation under FR light. During the dark-to-FR transition, phyB is rapidly imported into the nucleus and facilitates nuclear SPA1 accumulation. These findings support the notion that phyB plays a role in repressing FR light signaling. Activity modulation of the COP1-SPA E3 complex by light-activated phytochromes is an effective and pivotal regulatory step in light signaling.     

Activation of a Nodal-independent signaling pathway by Cripto-1 mutants with impaired activation of a Nodal-dependent signaling pathway

Cripto-1, a co-receptor for Nodal, can activate Nodal-dependent and Nodal-independent signaling pathways. In this study we have investigated whether Cripto-1 mutants, that fail to activate a Nodal-dependent signaling pathway, are capable to activate a Nodal-independent signaling pathway in mammary epithelial cells. Cripto-1 mutants expressed in EpH4 mouse mammary epithelial cells are fully functional in regard to activation of a Nodal-independent signaling pathway, leading to phosphorylation of mitogen-activated protein kinase (MAPK) and Akt and to enhanced proliferation and motility of these cells, suggesting that Cripto-1 mutants with impaired Nodal signaling are still active in a Nodal-independent signaling pathway.

Structured summary

MINT-6797299:

Glypican 1 (uniprotkb:P35052) physically interacts (MI:0218) with Cr 1 (uniprotkb:P13385) by anti bait coimmunoprecipitation (MI:0006)

     

Phospholipase D1 Regulates Lymphocyte Adhesion via Upregulation of Rap1 at the Plasma Membrane

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.Regulated adhesion of lymphocytes is required for immune function. The β2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates lymphocyte adhesion to endothelium, antigen-presenting cells, and virally infected target cells (14). These cell-cell adhesions enable lymphocyte trafficking in and out of lymphoid organs, T-cell activation, and cytotoxicity, respectively (2, 34). Thus, the regulation of LFA-1 adhesiveness is central to adaptive immunity.LFA-1 is a bidirectional receptor in that it mediates both outside-in and inside-out signaling (30). Outside-in signaling is analogous to signaling by conventional receptors and is defined as stimulation of intracellular signaling pathways as a consequence of ligation of LFA-1 with any of its extracellular ligands, such as intracellular adhesion molecule 1 (ICAM-1). Inside-out signaling refers to intracellular signaling events that result in a higher-affinity state of the ectodomain of LFA-1 for its cognate ligands. Regulatory events that mediate inside-out signaling converge on the cytoplasmic tails of the LFA-1 α and β chains, which transduce signals to their ectodomains (14). Signaling molecules implicated in inside-out signaling through LFA-1 include talin, Vav1, PKD1, several adaptor proteins (SLP-76, ADAP, and SKAP-55), the Ras family GTPase Rap1, and two of its effectors, RAPL and RIAM (26). How these proteins interact to activate LFA-1 remains poorly understood.Rap1 is a member of the Ras family of GTPases and has been implicated in growth control, protein trafficking, polarity, and cell-cell adhesion (6). The ability of activated Rap1 to promote LFA-1-mediated lymphocyte adhesion is well established (33). The physiologic relevance of this pathway is highlighted by leukocyte adhesion deficiency type III (LAD III), where immunocompromised patients have a congenital defect in GTP loading of Rap1 in leukocytes (24). LFA-1 is a plasma membrane protein, consistent with its role in cell-cell adhesion, which by definition is a cell surface phenomenon. Paradoxically, the bulk of Rap1 is expressed on intracellular vesicles. We have characterized these vesicles as recycling endosomes and have shown that the intracellular pool of Rap1 can be mobilized by exocytosis to augment the expression of Rap1 at the plasma membranes of lymphocytes, leading to increased adhesion (5). We used a fluorescent probe of activated Rap1 in live cells to show that only the pool of Rap1 at the plasma membrane becomes GTP bound upon lymphocyte activation. Thus, it appears that delivery of Rap1 via vesicular transport to the plasma membrane and activation of the GTPase on that compartment are linked. Among the signaling enzymes known to regulate vesicular trafficking is phospholipase D (PLD). Whereas PLD type 2 (PLD2) is expressed at the plasma membranes of lymphocytes, PLD1 is expressed on intracellular vesicles (29). We now show that PLD1 resides on the same vesicles as Rap1, is delivered along with Rap1 to the plasma membranes of stimulated T cells, and is required for Rap1 activation and T-cell adhesion.     

Therapeutic targeting of the thrombospondin-1 receptor CD47 to treat liver cancer

CD47 is a signaling receptor for the matricellular protein thrombospondin-1 and a counter-receptor for signal regulatory protein-α (SIRPα) on macrophages. Following its initial discovery in 1992 as a cell surface protein that is over-expressed by ovarian carcinoma, elevated CD47 expression has emerged as a negative prognostic factor for a variety of cancers. CD47 is also a potential therapeutic target based on the ability of CD47 blockade to cause regression of tumors in mice, and a humanized CD47 antibody has recently entered phase I clinical trials. CD47 blockade may control tumor growth by inhibiting thrombospondin-1 signaling or by preventing inhibitory SIRPα signaling in tumor-associated macrophages. A recent publication by Lee et al. (Hepatology 60:179–191, 2014) provides evidence that blocking CD47 signaling specifically depletes tumor-initiating stem cells in hepatocellular carcinoma and implicates cathepsin-S/protease-activated receptor-2 signaling in mediating this therapeutic response.     

Plasma membrane rafts and chaperones in cytokine/STAT signaling

We and others have recently obtained data suggesting that cytokine-STAT signaling in many different cell-types is a chaperoned pathway initiated at the level of specialized plasma membrane microdomains called "rafts" (the "raft-STAT signaling hypothesis"). These findings are of broad significance in that all cytokines and growth factors initiate signaling in target cells by interacting with respective cell-surface receptors. The new data suggest that raft microdomains represent the units of function at the cell-surface through which ligand-stimulated STAT signaling is initiated. Moreover, recent evidence shows the involvement of chaperone proteins in regulating the STAT signaling pathway. These chaperones include the human homolog of the tumorous imaginal disc 1 protein (hTid1) which associates with Janus kinase 2 (JAK2) at the level of the plasma membrane, heat shock protein 90 (HSP90) which associates with STAT3 and STAT1 proteins in caveolin-1-containing raft and cytoplasmic complexes, and glucose regulated protein 58 (GRP58/ER-60/ERp57), a thiol dependent protein-disulfide isomerase, found in association with STAT3 "statosome" complexes in the cytosol and in the raft fraction. We suggest a function of the HSP90 chaperone system in preserving IL-6/STAT3 signaling in liver cells in the context of fever. The identification and function of protein partners associated with specific STAT species in rafts and in cytosolic complexes, and in the efficient departure of cytokine-activated STATs from the cytosolic face of rafts towards the cell nucleus are now areas of active investigation.     

Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.Receptor tyrosine kinase (RTK) signaling is initiated on binding of soluble growth factors to growth factor receptors such as the insulin receptor (IR) or epidermal growth factor receptor (EGFR), or on binding of membrane-bound ephrins, as is the case for Eph receptors. Intracellular signaling is then propagated through PTMs, which commonly serve to regulate protein function by acting as docking sites for recruitment of modular protein interaction domains. Phosphorylation is the best studied PTM, and is a principle mechanism regulating intracellular signaling.A common element in RTK signaling involves autophosphorylation of the intracellular portion of the receptor (Fig. 1). RTKs become activated as a result of ligand-stabilized dimerization or oligomerization. For instance, in the EGFR subfamily (which includes ErbB and EGF receptors), the formation of homo- or heterodimers is initiated by ligand binding and subsequent exposure of a dimerization domain (Hynes and Lane 2005). Dimerization of the RTKs allows autophosphorylation of the RTKs; EGFR is exceptional in that an allosteric interaction between the kinase domains of adjacent monomers is responsible for the receptor activation (Zhang et al. 2006). However, in the majority of cases dimerization enhances RTK catalytic activity through phosphorylation of the kinase activation loop, and in some instances the juxtamembrane region, and recruits signaling effectors through the creation of pTyr docking sites. The specific interaction of signaling proteins with these pTyr-binding motifs activates signaling pathways, such as canonical signaling through the Ras-mitogen activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K)-Akt, and phospholipase C-gamma (PLC-γ) pathways. These RTK pathways can result in a variety of cellular processes, including differentiation, proliferation, survival, and migration (Fig. 1). The cellular context of signaling can dictate the biological outcome, and how each RTK initiates a given cellular process remains an area of active research.Open in a separate windowFigure 1.Receptor tyrosine kinases activate downstream pathways through recruitment of proteins containing pTyr-binding domains. Receptor tyrosine kinases are activated on growth factor binding to the extracellular domain of the receptor, leading to receptor dimerization and tyrosine phosphorylation (yellow circles labeled with a P) of their cytoplasmic tails, which act as docking sites for recruitment of PTB and SH2 domains. Various RTKs can mediate a diverse set of cellular processes (colored boxes) determined by the recruitment of specific SH2- and PTB-domain-containing proteins. The gray box displays how the adaptor Grb2 is recruited to an RTK through recognition of the pY-x-N (pY = pTyr, x = any natural amino acid) and activates cell growth and survival pathways such as MAPK and AKT, respectively, through complex formation via its SH3 domains.Tyrosine phosphorylation mediates RTK signaling through the recruitment and activation of proteins involved in downstream signaling pathways, mediated through pTyr binding of the SH2 and PTB domains of signaling effectors. SH2 and PTB domains are found in an otherwise diverse set of proteins containing a range of distinct catalytic and interaction domains, and provide a degree of specificity through their recognition of both a pTyr residue and surrounding amino acids. Here we will discuss the properties of proteins that contain SH2 and PTB domains and their roles in signaling downstream of RTKs, as well as the mechanisms by which they regulate the activity of these signaling effectors.     

The Ubiquitin Ligase PUB22 Targets a Subunit of the Exocyst Complex Required for PAMP-Triggered Responses in Arabidopsis

Plant pathogens are perceived by pattern recognition receptors, which are activated upon binding to pathogen-associated molecular patterns (PAMPs). Ubiquitination and vesicle trafficking have been linked to the regulation of immune signaling. However, little information exists about components of vesicle trafficking involved in immune signaling and the mechanisms that regulate them. In this study, we identified Arabidopsis thaliana Exo70B2, a subunit of the exocyst complex that mediates vesicle tethering during exocytosis, as a target of the plant U-box–type ubiquitin ligase 22 (PUB22), which acts in concert with PUB23 and PUB24 as a negative regulator of PAMP-triggered responses. We show that Exo70B2 is required for both immediate and later responses triggered by all tested PAMPs, suggestive of a role in signaling. Exo70B2 is also necessary for the immune response against different pathogens. Our data demonstrate that PUB22 mediates the ubiquitination and degradation of Exo70B2 via the 26S Proteasome. Furthermore, degradation is regulated by the autocatalytic turnover of PUB22, which is stabilized upon PAMP perception. We therefore propose a mechanism by which PUB22-mediated degradation of Exo70B2 contributes to the attenuation of PAMP-induced signaling.     

Selective regulation by delta-PKC and PI 3-kinase in the assembly of the antiapoptotic TNFR-1 signaling complex in neutrophils

TNF is implicated in the attenuation of neutrophil constitutive apoptosis during sepsis. Antiapoptotic signaling is mediated principally through the TNF receptor-1 (TNFR-1). In adherent neutrophils, when -integrin signaling is activated, TNF phosphorylates TNFR-1 and activates prosurvival and antiapoptotic signaling. Previously, we identified the -PKC isotype and phosphatidylinositol (PI) 3-kinase as critical regulators of TNF signaling in adherent neutrophils. Both kinases associate with TNFR-1 in response to TNF and are required for TNFR-1 serine phosphorylation, NF-B activation, and inhibition of apoptosis. The purpose of this study was to examine the role of -PKC and PI 3-kinase in the assembly of TNFR-1 signaling complex that regulates NF-B activation and antiapoptotic signaling. Coimmunoprecipitation studies established that PI 3-kinase, -PKC, and TNFR-1 formed a signal complex in response to TNF. -PKC recruitment required both -PKC and PI 3-kinase activity, whereas PI 3-kinase recruitment was -PKC independent, suggesting that PI 3-kinase acts upstream of -PKC. An important regulatory step in control of antiapoptotic signaling is the assembly of the TNFR-1-TNFR-1-associated death domain protein (TRADD)-TNFR-associated factor 2 (TRAF2)-receptor interacting protein (RIP) complex that controls NF-B activation. Inhibition of either -PKC or PI 3-kinase decreased TNF-mediated recruitment of RIP and TRAF2 to TNFR-1. In contrast, TRADD recruitment was enhanced. Thus -PKC and PI 3-kinase are positive regulators of TNF-mediated association of TRAF2 and RIP with TNFR-1. Conversely, these kinases are negative regulators of TRADD association. These results suggest that -PKC and PI 3-kinase regulate TNF antiapoptotic signaling at the level of the TNFR-1 through control of assembly of a TNFR-1-TRADD-RIP-TRAF2 complex. inflammation; tumor necrosis factor receptor-1-associated death domain protein; receptor interacting protein; tumor necrosis factor receptor-associated factor 2; antiapoptotic signaling     

SIRT1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling

Background

The directed differentiation of mesenchymal stem cells (MSCs) is tightly controlled by a complex network. Wnt signaling pathways have an important function in controlling the fate of MSCs. However, the mechanism through which Wnt/β-catenin signaling is regulated in differentiation of MSCs remains unknown. SIRT1 plays an important role in the regulation of MSCs differentiation.

Results

This study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells. First, the MSC commitment and differentiation model was established by using 5-azacytidine. Using the established model, C3H10T1/2 cells were treated with SIRT1 activator/inhibitor during differentiation. The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation. Notably, during commitment, resveratrol blocked adipocyte formation and promoted myotubes differentiation, whereas nicotinamide enhanced adipogenic potential of C3H10T1/2 cells. Furthermore, resveratrol elevated the expression of Cyclin D1 and β-catenin in the early stages. The luciferase assay showed that knockdown SIRT1 inhibits Wnt/β-catenin signaling, while resveratrol treatment or overexpression SIRT1 activates Wnt/β-catenin signaling. SIRT1 suppressed the expression of Wnt signaling antagonists sFRP2 and DACT1. Knockdown SIRT1 promoted adipogenic potential of C3H10T1/2 cells, whereas overexpression SIRT1 inhibited adipogenic differentiation and promoted myogenic differentiation.

Conclusions

Together, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

     

A Vertebrate-Specific Chp-PAK-PIX Pathway Maintains E-Cadherin at Adherens Junctions during Zebrafish Epiboly

Background

In early vertebrate development, embryonic tissues modulate cell adhesiveness and acto-myosin contractility to correctly orchestrate the complex processes of gastrulation. E-cadherin (E-cadh) is the earliest expressed cadherin and is needed in the mesendodermal progenitors for efficient migration [1], [2]. Regulatory mechanisms involving directed E-cadh trafficking have been invoked downstream of Wnt11/5 signaling [3]. This non-canonical Wnt pathway regulates RhoA-ROK/DAAM1 to control the acto-myosin network. However, in this context nothing is known of the intracellular signals that participate in the correct localization of E-cadh, other than a need for Rab5c signaling [3].

Methodology/Principal Findings

By studying loss of Chp induced by morpholino-oligonucleotide injection in zebrafish, we find that the vertebrate atypical Rho-GTPase Chp is essential for the proper disposition of cells in the early embryo. The underlying defect is not leading edge F-actin assembly (prominent in the cells of the envelope layer), but rather the failure to localize E-cadh and β-catenin at the adherens junctions. Loss of Chp results in delayed epiboly that can be rescued by mRNA co-injection, and phenocopies zebrafish E-cadh mutants [4], [5]. This new signaling pathway involves activation of an effector kinase PAK, and involvement of the adaptor PAK-interacting exchange factor PIX. Loss of signaling by any of the three components results in similar underlying defects, which is most prominent in the epithelial-like envelope layer.

Conclusions/Significance

Our current study uncovers a developmental pathway involving Chp/PAK/PIX signaling, which helps co-ordinate E-cadh disposition to promote proper cell adhesiveness, and coordinate movements of the three major cell layers in epiboly. Our data shows that without Chp signaling, E-cadh shifts to intracellular vesicles rather than the adhesive contacts needed for directed cell movement. These events may mirror the requirement for PAK2 signaling essential for the proper formation of the blood-brain barrier [6], [7].