LAPTM5 Protein Is a Positive Regulator of Proinflammatory Signaling Pathways in Macrophages

LAPTM5 (lysosomal-associated protein transmembrane 5) is a protein that is preferentially expressed in immune cells, and it interacts with the Nedd4 family of ubiquitin ligases. Recent studies in T and B cells identified LAPTM5 as a negative regulator of T and B cell receptor levels at the plasma membrane. Here we investigated the function of LAPTM5 in macrophages. We demonstrate that expression of LAPTM5 is required for the secretion of proinflammatory cytokines in response to Toll-like receptor ligands. We also show that RAW264.7 cells knocked down for LAPTM5 or macrophages from LAPTM5(-/-) mice exhibit reduced activation of NF-κB and MAPK signaling pathways mediated by the TNF receptor, as well as multiple pattern recognition receptors in various cellular compartments. TNF stimulation of LAPTM5-deficient macrophages leads to reduced ubiquitination of RIP1 (receptor-interacting protein 1), suggesting a role for LAPTM5 at the receptor-proximate level. Interestingly, we find that macrophages from LAPTM5(-/-) mice display up-regulated levels of A20, a ubiquitin-editing enzyme responsible for deubiquitination of RIP1 and subsequent termination of NF-κB activation. Our studies thus indicate that, in contrast to its negative role in T and B cell activation, LAPTM5 acts as a positive modulator of inflammatory signaling pathways and hence cytokine secretion in macrophages. They also highlight a role for the endosomal/lysosomal system in regulating signaling via cytokine and pattern recognition receptors.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

NGF controls dendrite development in hippocampal neurons by binding to p75NTR and modulating the cellular targets of Notch

Notch and neurotrophins control neuronal shape, but it is not known whether their signaling pathways intersect. Here we report results from hippocampal neuronal cultures that are in support of this possibility. We found that low cell density or blockade of Notch signaling by a soluble Delta-Fc ligand decreased the mRNA levels of the nuclear targets of Notch, the homologues of enhancer-of-split 1 and 5 (Hes1/5). This effect was associated with enhanced sprouting of new dendrites or dendrite branches. In contrast, high cell density or exposure of low-density cultures to NGF increased the Hes1/5 mRNA, reduced the number of primary dendrites and promoted dendrite elongation. The NGF effects on both Hes1/5 expression and dendrite morphology were prevented by p75-antibody (a p75NTR-blocking antibody) or transfection with enhancer-of-split 6 (Hes6), a condition known to suppress Hes activity. Nuclear translocation of NF-kappaB was identified as a link between p75NTR and Hes1/5 because it was required for the up-regulation of these two genes. The convergence of the Notch and p75NTR signaling pathways at the level of Hes1/5 illuminates an unexpected mechanism through which a diffusible factor (NGF) could regulate dendrite growth when cell-cell interaction via Notch is not in action.     

TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells

Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to derivatives of all three primary germ layers, yet little is known about the signaling cascades that govern their pluripotent character. Because it plays a prominent role in the early cell fate decisions of embryonic development, we have examined the role of TGFbeta superfamily signaling in hESCs. We found that, in undifferentiated cells, the TGFbeta/activin/nodal branch is activated (through the signal transducer SMAD2/3) while the BMP/GDF branch (SMAD1/5) is only active in isolated mitotic cells. Upon early differentiation, SMAD2/3 signaling is decreased while SMAD1/5 signaling is activated. We next tested the functional role of TGFbeta/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extend these findings to show that SMAD2/3 activation is required downstream of WNT signaling, which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly, we show that in ex vivo mouse blastocyst cultures, SMAD2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a crucial role for TGFbeta signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFbeta and WNT signaling in these contexts.     

Stretch-induced phosphorylation of focal adhesion kinase in endothelial cells: role of mitochondrial oxidants

Mechanical stretch activates a number of signaling pathways in endothelial cells, and it elicits a variety of functional responses including increases in the phosphorylation of focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin-mediated signal transduction. Stretch also triggers an increase in the generation of reactive oxygen species (ROS), which may function as second messengers in the signal transduction cascades that activate cellular responses to strain. Mitochondria represent an important source of ROS in the cell, and these organelles may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. We therefore tested whether cyclic stretch increases FAK phosphorylation at Tyr397 through a mitochondrial ROS signaling pathway in bovine pulmonary artery endothelial cells (BPAEC). Oxidant signaling, measured using 2'7'-dichlorofluorescin (DCFH), increased 152 +/- 16% during 1.5 h of cyclic strain relative to unstrained controls. The mitochondrial inhibitors diphenylene iodonium (5 microM) or rotenone (2 microM) attenuated this increase, whereas L-nitroarginine (100 microM), allopurinol (100 microM), or apocynin (30 microM) had no effect. The antioxidants ebselen (5 microM) and dithiodidiethyldithiocarbamate (1 mM) inhibited the strain-induced increase in oxidant signaling, but Hb (5 microM) had no effect. These results indicate that strain induces oxidant release from mitochondria. Treatment with cytochalasin D (5 microM) abrogated strain-induced DCFH oxidation in BPAEC, indicating that actin filaments were required for stretch-induced mitochondrial ROS generation. Cyclic strain increased FAK phosphorylation at Tyr397, but this was abolished by mitochondrial inhibitors as well as by antioxidants. Strain-induced FAK phosphorylation was abrogated by inhibition of protein kinase C (PKC) with Ro-31-8220 or G?-6976. These findings indicate that mitochondrial oxidants generated in response to endothelial strain trigger FAK phosphorylation through a signaling pathway that involves PKC.     

The loss of glypican-3 induces alterations in Wnt signaling

Loss-of-function mutations of the GPC3 gene are the cause of the human Simpson-Golabi-Behmel syndrome. Based on the overgrowth phenotype of the Simpson-Golabi-Behmel syndrome patients and the key role played by the insulin-like growth factor (IGF) signaling system in regulating embryonic growth, it was speculated that GPC3 regulates IGF signaling. In order to test the validity of this hypothesis, we mated GPC3 knockout mice with insulin receptor substrate-1 (IRS-1) nullizygous mice. We found that GPC3 regulates organism growth independent of IRS-1, suggesting that GPC3 does not modulate IGF signaling. Instead, we found that GPC3 knockout mice exhibit alterations in the Wnt signaling pathway, which is also associated with the regulation of cell proliferation. In particular, the loss of GPC3 led to the inhibition of the non-canonical Wnt/JNK signaling pathway, while concomitantly causing the activation of canonical Wnt/beta-catenin signaling. These in vivo findings were confirmed in vitro upon the ectopic overexpression of GPC3 in mesothelioma cells. In these cells, the GPC3-induced increase in JNK activity was associated with an enhanced response to Wnt5a. Most interestingly, the heparan sulfate chains of GPC3 were not required for its stimulatory activity on Wnt5a signaling and for the formation of GPC3-Wnt5a complexes. We propose that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling, by potentiating non-canonical Wnt signaling, while inhibiting the canonical Wnt signaling pathway.     

Membrane localization of scaffold proteins promotes graded signaling in the yeast MAP kinase cascade

BACKGROUND: Signaling through mitogen-activated protein kinase (MAPK) cascade pathways can show various input-output behaviors, including either switch-like or graded responses to increasing levels of stimulus. Prior studies suggest that switch-like behavior is promoted by positive feedback loops and nonprocessive phosphorylation reactions, but it is unclear whether graded signaling is a default behavior or whether it must be enforced by separate mechanisms. It has been hypothesized that scaffold proteins promote graded behavior. RESULTS: Here, we experimentally probe the determinants of graded signaling in the yeast mating MAPK pathway. We find that graded behavior is robust in that it resists perturbation by loss of several negative-feedback regulators. However, the pathway becomes switch-like when activated by a crosstalk stimulus that bypasses multiple upstream components. To dissect the contributing factors, we developed a method for gradually varying the signal input at different pathway steps in vivo. Input at the beginning of the kinase cascade produced a sharp, threshold-like response. Surprisingly, the scaffold protein Ste5 increased this threshold behavior when limited to the cytosol. However, signaling remained graded whenever Ste5 was allowed to function at the plasma membrane. CONCLUSIONS: The results suggest that the MAPK cascade module is inherently ultrasensitive but is converted to a graded system by the pathway-specific activation mechanism. Scaffold-mediated assembly of signaling complexes at the plasma membrane allows faithful propagation of weak signals, which consequently reduces pathway ultrasensitivity. These properties help shape the input-output properties of the system to fit the physiological context.     

A Positive Feedback Loop Involving Gcm1 and Fzd5 Directs Chorionic Branching Morphogenesis in the Placenta

Chorioallantoic branching morphogenesis is a key milestone during placental development, creating the large surface area for nutrient and gas exchange, and is therefore critical for the success of term pregnancy. Several Wnt pathway molecules have been shown to regulate placental development. However, it remains largely unknown how Wnt-Frizzled (Fzd) signaling spatiotemporally interacts with other essential regulators, ensuring chorionic branching morphogenesis and angiogenesis during placental development. Employing global and trophoblast-specific Fzd5-null and Gcm1-deficient mouse models, combining trophoblast stem cell lines and tetraploid aggregation assay, we demonstrate here that an amplifying signaling loop between Gcm1 and Fzd5 is essential for normal initiation of branching in the chorionic plate. While Gcm1 upregulates Fzd5 specifically at sites where branching initiates in the basal chorion, this elevated Fzd5 expression via nuclear β-catenin signaling in turn maintains expression of Gcm1. Moreover, we show that Fzd5-mediated signaling induces the disassociation of cell junctions for branching initiation via downregulating ZO-1, claudin 4, and claudin 7 expressions in trophoblast cells at the base of the chorion. In addition, Fzd5-mediated signaling is also important for upregulation of Vegf expression in chorion trophoblast cells. Finally, we demonstrate that Fzd5-Gcm1 signaling cascade is operative during human trophoblast differentiation. These data indicate that Gcm1 and Fzd5 function in an evolutionary conserved positive feedback loop that regulates trophoblast differentiation and sites of chorionic branching morphogenesis.     

ERalpha and STAT5a cross-talk: interaction through C-terminal portions of the proteins decreases STAT5a phosphorylation, nuclear translocation and DNA-binding

Cross-talk between ERalpha and STAT5a was demonstrated to mediate through a direct physical association between the two proteins. By GST pull-down assays and functional assays with various constructs of ERalpha and STAT5a, it was shown that the C-termini of these two proteins were mainly responsible for this interaction. Furthermore, the interaction between ERalpha and STAT5a was demonstrated to give rise to functional changes in their signaling events. In cell transfection studies, it was shown that ERalpha activation could attenuate PRLR signaling through STAT5a. This ERalpha-mediated attenuation of PRLR signaling was substantiated by observed decreases in the phosphorylation of JAK2 and STAT5a, reduced translocation of STAT5a into the nucleus, and reduced binding of STAT5a onto a GAS-containing nucleotide. Apart from transfected cells, the interaction between ERalpha and STAT5a could also be observed in established breast cancer cell lines of MCF-7 and T-47D in co-immunoprecipitation studies. However, the functional consequence of the interaction in these cancer cells was very different from the transfected HEK293 cells. ER activation could lead to potentiation of PRLR signaling in MCF-7 cells but not in T-47D cells. Conversely, in both MCF-7 and T-47D cells, PRLR activation could lead to attenuation of ER signaling. These data serve to elucidate the mechanisms underlying the ERalpha-STAT5a cross-talk and in demonstrating that the functional consequence of this cross-talk depends on the precise milieus of the intracellular environment.     

Lineage-specific negative regulation of STAT-mediated signaling by proteolytic processing

Accumulating evidence suggests that STAT-mediated signaling plays critical roles in cell differentiation and/or cell expansion and that in turn, STAT-mediated signaling is regulated strictly by many mechanisms. In murine mast cells, when Stat6 is activated by IL-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease (namely Stat6-protease). Similarly, the activated Stat5 is cleaved by a protease (Stat5-protease) in the nucleus of myeloid progenitors. These STAT proteases cleave the corresponding STAT proteins at the carboxyl-terminus and the resultant STAT proteins function as dominant negative molecules. Functionally, Stat6-protease protects mast cells from Stat6-dependent growth inhibition while Stat5-protease maintains the immature state of myeloid progenitors. In addition, it has been shown that the activated Stat3 is cleaved in mature neutrophils. These findings indicate that the proteolytic processing of STAT proteins by the nucleus-associated protease functions as a lineage-specific negative-regulator of STAT-mediated signaling.     

Tissue transglutaminase clusters soluble A-type ephrins into functionally active high molecular weight oligomers

The Eph receptors and their ligands, the ephrins, are thought to act at points of close cell-cell contact to elicit bi-directional signaling in receptor and ligand expressing cells. However, when cultured in vitro, some A-type ephrins are released from the cell surface and it is unclear if these soluble ephrins participate in Eph receptor activation. We show that soluble ephrin A5 is subject to oligomerization. Ephrins A1 and A5 are substrates for a cross-linking enzyme, tissue transglutaminase, which mediates the formation of oligomeric ephrin. Transglutaminase-cross-linked ephrin binds to A-type Eph receptors, stimulates Eph kinase activity, and promotes invasion and migration of HeLa cells. Transglutaminase-mediated oligomerization of soluble ephrin potentially represents a novel mechanism of forward signaling through Eph receptors and may extend the influence of A-type ephrins beyond cell contact mediated signaling.     

The INAD scaffold is a dynamic, redox-regulated modulator of signaling in the Drosophila eye

INAD is a scaffolding protein that regulates signaling in Drosophila photoreceptors. One of its PDZ domains, PDZ5, cycles between reduced and oxidized forms in response to light, but it is unclear how light affects its redox potential. Through biochemical and structural studies, we show that the redox potential of PDZ5 is allosterically regulated by its interaction with another INAD domain, PDZ4. Whereas isolated PDZ5 is stable in the oxidized state, formation of a PDZ45 "supramodule" locks PDZ5 in?the reduced state by raising the redox potential of?its Cys606/Cys645 disulfide bond by ～330?mV. Acidification, potentially mediated via light and PLCβ-mediated hydrolysis of PIP(2), disrupts the interaction between PDZ4 and PDZ5, leading to PDZ5 oxidation and dissociation from the TRP Ca(2+) channel, a key component of fly visual signaling. These results show that scaffolding proteins can actively modulate the intrinsic redox potentials of their disulfide bonds to exert regulatory roles in signaling.     

Anaphylatoxin signaling in human neutrophils. A key role for sphingosine kinase

Anaphylatoxins activate immune cells to trigger the release of proinflammatory mediators that can lead to the pathology of several immune-inflammatory diseases. However, the intracellular signaling pathways triggered by anaphylatoxins are not well understood. Here we report for the first time that sphingosine kinase (SPHK) plays a key role in C5a-triggered signaling, leading to physiological responses of human neutrophils. We demonstrate that C5a rapidly stimulates SPHK activity in neutrophils and differentiated HL-60 cells. Using the SPHK inhibitor N,N-dimethylsphingosine (DMS), we show that inhibition of SPHK abolishes the Ca2+ release from internal stores without inhibiting phospholipase C or protein kinase C activation triggered by C5a but has no effect on calcium signals triggered by other stimuli (FcgammaRII). We also show that DMS inhibits degranulation, activation of the NADPH oxidase, and chemotaxis triggered by C5a. Moreover, an antisense oligonucleotide against SPHK1, in neutrophil-differentiated HL-60 cells, had similar inhibitory properties as DMS, suggesting that the SPHK utilized by C5a is SPHK1. Our data indicate that C5a stimulation decreases cellular sphingosine levels and increases the formation of sphingosine-1-phosphate. Exogenously added sphingosine has a dual effect on C5a-stimulated oxidative burst: it has a priming effect at lower concentrations but a dose-dependent inhibitory effect at higher concentrations; however, C5a-triggered protein kinase C activity was only reduced at high concentration of sphingosine. In contrast, C5a-triggered Ca2+ signals, chemotaxis, and degranulation were not affected by sphingosine at all. Exogenous sphingosine-1-phosphate, by itself, did not induce degranulation or chemotaxis, but it did marginally induce Ca2+ signals and oxidative burst and had a priming effect, enhancing all the C5a-triggered responses. Taken together, these results suggest that SPHK plays an important role in the immune-inflammatory pathologies triggered by anaphylatoxins in human neutrophils and point out SPHK as a potential therapeutic target for the treatment of diseases associated with neutrophil hyperactivation.     

Multiple signal transduction pathways mediated by 5-HT receptors

Among human serotonin (5-HT) receptor subtypes, each G protein-coupled receptor subtype is reported to have one G protein-signaling cascade. However, the signaling may not be as simple as previously thought to be. 5-HT5A receptors are probably the least well understood among the 5-HT receptors, but the authors found that 5-HT5A receptors couple to multiple signaling cascades. When the 5-HT5A receptors were expressed in undifferentiated C6 glioma cells, they modulated the level of second messengers. For example, activation of 5-HT5A receptors inhibited the adenylyl cyclase activity and subsequently reduced the cAMP level, as previously reported. In addition to this known signaling via Gi/Go, 5-HT5A receptors are coupled to the inhibition of ADP-ribosyl cyclase and cyclic ADP ribose formation. On the other hand, activation of 5-HT5A receptors transiently opened the K+ channels, presumably due to the increase in intracellular Ca2+ after formation of inositol (1,4,5) trisphosphate. The K+ currents were inhibited by both heparin and pretreatment with pertussis toxin, suggesting the cross-talk between Gi/Go protein and phopholipase C cascade. Thus, the authors results indicate that 5-HT5A receptors couple to multiple second messenger systems and may contribute to the complicated physiological and pathophysiological states. Although this multiple signaling has been reported only for 5-HT5A/5-HT1 receptors so far, it is possible that other 5-HT receptor subtypes bear similar complexity. As a result, in addition to the wide variety of expression patterns of each 5-HT receptor subtype, it is possible that multiple signal transduction systems may add complexity to the serotonergic system in brain function. The investigation of these serotonergic signaling and its impairment at cellular level may help to understand the symptoms of brain diseases.     

Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis

Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components.