Receptor-independent Signaling Research Articles

Force-control at cellular membranes.

Force-regulation at cellular membranes relies on dynamic molecular platforms that integrate intra- and extracellular signals to control cell shape and function. To correctly respond to a continuously changing environment, activity of these platforms needs to be tightly controlled in space and time. Over the last few years, curvature-dependent mechano-chemical signal translation—a receptor-independent signaling mechanism where physical forces at the plasma membrane trigger nanoscale membrane deformations that are then translated into chemical signal transduction cascades—has emerged as a new signaling principle that cells use to regulate forces at the membrane. However, until recently, technical limitations have precluded studies of this force-induced curvature-dependent signaling at the physiological scale. Here, we comment on recent advancements that allow studying curvature-dependent signaling at membranes, and discuss processes where it may be involved in. Considering its general impact on cell function, a particular focus will be put on the curvature-dependence of feedback loops that control actin-based forces at cellular membranes.

Rictor negatively regulates high-affinity receptors for IgE-induced mast cell degranulation.

Rictor is a regulatory component of the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). We have previously demonstrated that rictor expression is substantially downregulated in terminally differentiated mast cells as compared with their immature or transformed counterparts. However, it is not known whether rictor and mTORC2 regulate mast cell activation. In this article, we show that mast cell degranulation induced by aggregation of high-affinity receptors for IgE (FcεRI) is negatively regulated by rictor independently of mTOR. We found that inhibition of mTORC2 by the dual mTORC1/mTORC2 inhibitor Torin1 or by downregulation of mTOR by short hairpin RNA had no impact on FcεRI-induced degranulation, whereas downregulation of rictor itself resulted in an increased sensitivity (∼50-fold) of cells to FcεRI aggregation with enhancement of degranulation. This was linked to a similar enhancement in calcium mobilization and cytoskeletal rearrangement attributable to increased phosphorylation of LAT and PLCγ1. In contrast, degranulation and calcium responses elicited by the G protein-coupled receptor ligand, C3a, or by thapsigargin, which induces a receptor-independent calcium signal, was unaffected by rictor knockdown. Overexpression of rictor, in contrast with knockdown, suppressed FcεRI-mediated degranulation. Taken together, these data provide evidence that rictor is a multifunctional signaling regulator that can regulate FcεRI-mediated degranulation independently of mTORC2.

Death receptor-independent FADD signalling triggers hepatitis and hepatocellular carcinoma in mice with liver parenchymal cell-specific NEMO knockout.

Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO(LPC-KO) mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO(LPC-KO) mice. To address potential functional redundancies between death receptors we generated and analysed NEMO(LPC-KO) mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO(LPC-KO) mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO(LPC-KO) mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO(LPC-KO) mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.

BB2 bombesin receptor-expressing spinal neurons transmit herpes-associated itch by BB2 receptor-independent signaling

Although spinal neurons expressing BB2 bombesin receptors are suggested to be involved in itch transmission, their role in pathological itch remains unknown. Because itch is often observed in patients with herpes zoster, we examined the role of BB2 receptor-expressing spinal neurons in herpes-associated itch in mice. Transdermal inoculation of human herpes virus 1 on the midflank produced herpes zoster-like skin lesions and caused the mice to scratch (itch-related behavior) and lick (pain-related behavior) the affected skin. Ablation of BB2 receptor-expressing spinal neurons by intrathecal treatment with a bombesin-saporin conjugate decreased the scratching but not the licking. Intrathecal administration of the BB2 receptor antagonist Leu13-ψ-(CH2NH)Leu14-bombesin decreased BB2 receptor agonist GRP(18-27)-induced scratching in naive mice but not herpes-associated scratching. The present results suggest that BB2 receptor-expressing spinal neurons transmit herpes-associated itch by BB2 receptor-independent signaling.

Dynamic Motions of G Proteins during Receptor Independent Cell Signaling

The Gα subunit is responsible for regulating intracellular signals through guanine nucleotide binding and release, the catalytic rate limiting step. Recent studies have shown interactions of Gα with Guanine nucleotide Exchange Factors (GEFs) cause dynamic structural changes which facilitate substrate release. Determining the structural changes and dynamics of the GDP-bound (inactive) and GEF-bound (nucleotide-free) states of the α-subunit will lead to a better understanding of cell signaling and regulation. In this study the conformational state distributions and dynamic motions of Gα were probed on a surface-cysteine free Gαi1 construct (Gα Hexa I) mutant using single molecule Forster Resonance Energy Transfer (smFRET)and time-resolved fluorescence anisotropy.

Role of Phospholipid Oxidation Products in Atherosclerosis

There is increasing clinical evidence that phospholipid oxidation products (Ox-PL) play a role in atherosclerosis. This review focuses on the mechanisms by which Ox-PL interact with endothelial cells, monocyte/macrophages, platelets, smooth muscle cells, and HDL to promote atherogenesis. In the past few years major progress has been made in identifying these mechanisms. It has been recognized that Ox-PL promote phenotypic changes in these cell types that have long-term consequences for the vessel wall. Individual Ox-PL responsible for specific cellular effects have been identified. A model of the configuration of bioactive truncated Ox-PL within membranes has been developed that demonstrates that the oxidized fatty acid moiety protrudes into the aqueous phase, rendering it accessible for receptor recognition. Receptors and signaling pathways for individual Ox-PL species are now determined and receptor independent signaling pathways identified. The effects of Ox-PL are mediated both by gene regulation and transcription independent processes. It has now become apparent that Ox-PL affects multiple genes and pathways, some of which are proatherogenic and some are protective. However, at concentrations that are likely present in the vessel wall in atherosclerotic lesions, the effects promote atherogenesis. There have also been new insights on enzymes that metabolize Ox-PL and the significance of these enzymes for atherosclerosis. With the knowledge we now have of the regulation and effects of Ox-PL in different vascular cell types, it should be possible to design experiments to test the role of specific Ox-PL on the development of atherosclerosis.

Oncogenic KRAS Impairs EGFR Antibodies' Efficiency by C/EBPβ-Dependent Suppression of EGFR Expression

Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRAS(G12V)) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRAS(G12V) on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRAS(G12V) impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRAS(G12V) also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs-such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRAS(G12V) downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPβ-LIP. Thus, siRNA-mediated knockdown of C/EBPβ led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRAS(G12V) signaling induced C/EBPβ-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents.

Extrarenal effects of aldosterone

The renal distal tubule has been considered for a long time as the main cellular target of aldosterone, where the hormone enhances sodium reabsorption and potassium secretion. However, other cell types in nonepithelial tissues, such as the heart, the vessels, adipose tissue, and macrophages, are now also recognized as targets for aldosterone. The functions that aldosterone exerts in these nonclassical target tissues are still a matter of debate. This review will highlight the recent findings on the extrarenal effects of aldosterone. Numerous studies showed that aldosterone exerts profibrotic and proinflammatory effects, but one or more cofactors such as salt, angiotensin II, and oxidative stress are required. Moreover, inflammation and macrophage infiltration are a prerequisite to aldosterone-induced cardiac fibrosis. This underlines a key role for aldosterone and the mineralocorticoid receptor in macrophages. Inflammatory effects of aldosterone in vascular smooth muscle cells involve trafficking to lipid rafts/caveolae through receptor tyrosine kinases. Finally, a growing body of evidence indicates a prominent role of aldosterone/mineralocorticoid receptor in the metabolic syndrome, in insulin resistance, and in adipocyte biology. The idiom from Socrates, 'the more we learn, the less we know', can be applied to aldosterone with its different facets and its pleiotropic effects. There is clear evidence for rapid nongenomic effects of aldosterone, mineralocorticoid receptor-dependent and mineralocorticoid receptor-independent signaling, in the heart, the vessels, and other nonepithelial tissues, leading to inflammation, fibrosis, and progression of cardiovascular diseases including hypertension and metabolic syndrome.

Adjuvanticity of the oil-in-water emulsion MF59 is independent of Nlrp3 inflammasome but requires the adaptor protein MyD88

Oil-in-water emulsions have been successfully used to increase the efficacy, immunogenicity, and cross-protection of human vaccines; however, their mechanism of action is still largely unknown. Nlrp3 inflammasome has been previously associated to the activity of alum, another adjuvant broadly used in human vaccines, and MyD88 adaptor protein is required for the adjuvanticity of most Toll-like receptor agonists. We compared the contribution of Nlrp3 and MyD88 to the adjuvanticity of alum, the oil-in-water emulsion MF59, and complete Freund's adjuvant in mice using a three-component vaccine against serogroup B Neisseria meningitidis (rMenB). Although the basal antibody responses to the nonadjuvanted rMenB vaccine were largely dependent on Nlrp3, the high-level antibody responses induced by alum, MF59, or complete Freund's adjuvant did not require Nlrp3. Surprisingly, we found that MF59 requires MyD88 to enhance bactericidal antibody responses to the rMenB vaccine. Because MF59 did not activate any of the Toll-like receptors in vitro, we propose that MF59 requires MyD88 for a Toll-like receptor-independent signaling pathway.

Selective Small Molecule Inhibitors Of Heterotrimeric G‐Protein Signaling For The Treatment Of Ovarian Cancer

Overactive signaling at the level of hormone, receptor, or G‐protein can initiate and potentiate cellular transformation and other diseases. We hypothesize that direct inhibition of overactive G‐protein signaling is cytotoxic to ovarian cancers. We describe the development of selective small molecule inhibitors which bind to and stabilize the GDP‐bound form of the Galphai1. Initial inhibitors were identified using computational high throughput docking of a 280,000 small molecule database to Galphai1‐GDP and counter‐screened against Galphai1‐GTP and Galphaq‐GDP. Nucleotide exchange assays have verified selective G‐protein inhibition. This inhibition was supported by live cell bioluminescence resonance energy transfer (BRET) experiments measuring the interaction of Galphai1 with the regulatory protein AGS4 in HEK293 cells. Inhibition of Galphai1 but not Galphaq decreased cell viability in SKOV3 ovarian cancer cells. The mechanism of Gi pathway inhibition is also explored. Pharmacologic regulation of both receptor dependent and independent signaling through specific heterotrimeric subunits will provide a unique window into a major signaling axis while providing data and compounds for the treatment of a refractory disease like ovarian cancer.

TGF-β induces TIAF1 self-aggregation via type II receptor-independent signaling that leads to generation of amyloid β plaques in Alzheimer's disease

The role of a small transforming growth factor beta (TGF-β)-induced TIAF1 (TGF-β1-induced antiapoptotic factor) in the pathogenesis of Alzheimer's disease (AD) was investigated. TIAF1 physically interacts with mothers against DPP homolog 4 (Smad4), and blocks SMAD-dependent promoter activation when overexpressed. Accordingly, knockdown of TIAF1 by small interfering RNA resulted in spontaneous accumulation of Smad proteins in the nucleus and activation of the promoter governed by the SMAD complex. TGF-β1 and environmental stress (e.g., alterations in pericellular environment) may induce TIAF1 self-aggregation in a type II TGF-β receptor-independent manner in cells, and Smad4 interrupts the aggregation. Aggregated TIAF1 induces apoptosis in a caspase-dependent manner. By filter retardation assay, TIAF1 aggregates were found in the hippocampi of nondemented humans and AD patients. Total TIAF1-positive samples containing amyloid β (Aβ) aggregates are 17 and 48%, respectively, in the nondemented and AD groups, suggesting that TIAF1 aggregation occurs preceding formation of Aβ. To test this hypothesis, in vitro analysis showed that TGF-β-regulated TIAF1 aggregation leads to dephosphorylation of amyloid precursor protein (APP) at Thr668, followed by degradation and generation of APP intracellular domain (AICD), Aβ and amyloid fibrils. Polymerized TIAF1 physically interacts with amyloid fibrils, which would favorably support plaque formation in vivo.

Beneficial effects of cannabinoids (CB) in a murine model of allergen-induced airway inflammation: Role of CB 1/CB 2 receptors

The endocannabinoid system (ECS) consists of two cannabinoid (CB) receptors, namely CB 1 and CB 2 receptor, and their endogenous (endocannabinoids) and exogenous (cannabinoids, e.g. delta-9-tetrahydrocannabinol (THC)) ligands which bind to these receptors. Based on studies suggesting a role of THC and the ECS in inflammation, the objective of this study was to examine their involvement in type I hypersensitivity using a murine model of allergic airway inflammation. THC treatment of C57BL/6 wildtype mice dramatically reduced airway inflammation as determined by significantly reduced total cell counts in bronchoalveolar lavage (BAL). These effects were greatest when mice were treated during both, the sensitization and the challenge phase. Furthermore, systemic immune responses were significantly suppressed in mice which received THC during sensitization phase. To investigate a role of CB 1/2 receptors in this setting, we used pharmacological blockade of CB 1 and/or CB 2 receptors by the selective antagonists and moreover CB 1/CB 2 receptor double-knockout mice (CB 1 −/−/CB 2 −/−) and found neither significant changes in the cell patterns in BAL nor in immunoglobulin levels as compared to wildtype mice. Our results indicate that the activation of the ECS by applying the agonist THC is involved in the development of type I allergies. However, CB 1/CB 2 receptor-independent signalling seems likely in the observed results.

Novel pathway for an old neurotransmitter: Dopamine-induced neuronal calcium signalling via receptor-independent mechanisms

Dopamine is one of the key neurotransmitters in the central nervous system and plays an important role in physiological processes, as well as in the development of many diseases. Here we report a receptor-independent signalling pathway induced by dopamine in CNS neurons. In cultured neurons from midbrain, cortex and hippocampus, dopamine uptake via dopamine or monoamine transporters induces plasmalemmal membrane depolarization, leading to opening of voltage gated calcium channels and a cytosolic calcium signal. This dopamine-induced calcium signal is unaffected by inhibition of the known dopamine receptors. In anaesthetized rats, application of dopamine in the presence of dopamine receptor antagonists to brainstem structures controlling cardiovascular activity results in an increase in heart rate, arterial blood pressure and sympathetic nerve activity. These data identify a novel dopamine-induced signalling pathway in CNS neurons which may have an important functional role in the central mechanisms controlling complex behaviours.

Amphetamine up-regulates activator of G-protein signaling 1 mRNA and protein levels in rat frontal cortex: the role of dopamine and glucocorticoid receptors

Acute and chronic exposure to psychostimulants results in altered function of G-protein-coupled receptors in the forebrain. It is believed that neuroadaptations in G-protein signaling contribute to behavioral sensitivity to psychostimulants that persists over a prolonged drug-free period. Proteins termed activators of G-protein signaling (AGS) have been characterized as potent modulators of both receptor-dependent and receptor-independent G-protein signaling. Nevertheless, the regulation of AGS gene and protein expression by psychostimulants remains poorly understood. In the present study, we investigated amphetamine (AMPH)-induced changes in expression patterns of several forebrain-enriched AGS proteins. A single exposure to AMPH (2.5 mg/kg i.p.) selectively induced gene expression of AGS1, but not Rhes or AGS3 proteins, in the rat prefrontal cortex (PFC) as measured 3 h later. Induction of AGS1 mRNA in the PFC by acute AMPH was transient and dose-dependent. Even repeated treatment with AMPH for 5 days did not produce lasting changes in AGS1 mRNA and protein levels in the PFC as measured 3 weeks post treatment. However, at this time point, a low dose AMPH challenge (1 mg/kg i.p.) induced a robust behavioral response and upregulated AGS1 expression in the PFC selectively in animals with an AMPH history. The effects of AMPH on AGS1 expression in the PFC were blocked by a D2, but not D1, dopamine receptor antagonist and partially by a glucocorticoid receptor antagonist. Collectively, the present study suggests that (1) AGS1 represents a regulator of G-protein signaling that is rapidly inducible by AMPH in the frontal cortex, (2) AGS1 regulation in the PFC parallels behavioral activation by acute AMPH in drug-naive animals and hypersensitivity to AMPH challenge in sensitized animals, and (3) D2 dopamine and glucocorticoid receptors regulate AMPH effects on AGS1 in the PFC. Changes in AGS1 levels in the PFC may result in abnormal receptor-to-G-protein coupling that alters cortical sensitivity to psychostimulants.

A Subpopulation of Follicular Lymphoma Tumor Infiltrating T Cells Shows Suppressed Common Gamma Chain Cytokine Signaling.

Abstract 759 Introduction:Tumor infiltrating T cells present within biopsy specimens of human B cell non-Hodgkin's lymphomas (NHL) provide a valuable opportunity to examine immune system function in the presence of cancer. We recently used flow cytometry to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a novel lymphoma cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome. Here, we turned our attention to signaling differences in subsets of the tumor-infiltrating T cells from FL and two other NHLs, diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Signaling differences that distinguish the tumor infiltrating T cells from each malignancy might be expected to be a reflection of the specific disease microenvironment, whereas T cell signaling differences distinguishing cases of the same malignancy might be related to the biology of each patient's tumor. Methods:Single cell flow cytometry measurements of signaling were acquired for samples of DLBCL (N=13), MCL (N=20), and FL (N=14). Phosphorylation of 14 signaling proteins was measured under 12 stimulation conditions in every cell, including lymphoma B cells and tumor-infiltrating T cells within the same specimen. Stimulation conditions included those that were B cell specific (BCR crosslinking, CD40 ligand), T cell specific (IL-7), and those that stimulated both B and T cells (IL-4, IL-10, IL-21, PMA + ionomycin, and IFN-γ). Results:Striking differences were observed in the signaling responses of tumor infiltrating T cells. T cells infiltrating FL patient samples showed significantly lower responses to cytokines where signal transduction is mediated by the common γ chain receptor. Specifically, we observed significant lower induction of p-STAT6 after IL-4 stimulation, p-STAT5 after IL-7 stimulation, and p-STAT3 after IL-21 stimulation (p < 0.001 for FL vs. MCL in all cases). In contrast, receptor-independent signaling was not significantly different as FL tumor infiltrating T cells responded at a level comparable to MCL and DLBCL tumor infiltrating T cells when stimulated with PMA and ionomycin. The lower response to common γ chain family cytokines could be the result of a partial suppression of all tumor infiltrating T cells or a complete suppression of a distinct subset. To distinguish between these possibilities, we analyzed signaling in tumor infiltrating T cell subsets. This single cell approach showed that tumor infiltrating T cells were a heterogeneous mixture of non-responsive cells and highly responsive T cells in response to cytokines. Specifically, the mean percentage of T cells that did not induce p-STAT3 after IL-21 stimulation was 50.3% in FL samples in contrast to only 26.2% in MCL samples. Phenotypic analysis showed that the vast majority of T cells infiltrating FL patient samples were CD4+CD45RO memory cells, and the single cell signaling approach revealed that the FL nonresponsive T cell subset had this phenotype. Furthermore, FL T cells were composed of a significantly larger fraction of T regulatory cells than MCL T cells, on average 17% FoxP3+CD25+ cells compared to only 9% in MCL (p<0.0002). Experiments are ongoing to test whether the prevalence of T regulatory cells influence the signaling capacity of the remaining CD4 conventional T cells. Conclusions:A subpopulation of tumor infiltrating T cells within FL patient samples has reduced responsiveness to the common gamma chain family members IL-4, IL-7 and IL-21, and distinguishes FL from DLBCL and MCL. These results may reflect a more suppressive microenvironment in FL. Disclosures:No relevant conflicts of interest to declare.

Intracellular Serotonin Modulates Insulin Secretion from Pancreatic β-Cells by Protein Serotonylation

Author SummaryDiabetes is the most prevalent metabolic disease and one that affects individuals of every social and economic status. The disease can arise as a result of reduced secretion of insulin from pancreatic β-cells or reduced action of insulin on its target organs. Therefore, understanding how to prevent and treat diabetes requires an extensive knowledge of the regulation of insulin secretion. In this study, we identify the hormone serotonin as a new regulator of insulin secretion and thereby attribute a function to the co-localization of serotonin and insulin in pancreatic β-cells that was first observed 30 years ago but until now not understood. We first demonstrate that a lack of serotonin in β-cells of transgenic mice leads to reduced insulin secretion and diabetes mellitus and that pharmacological replenishment of serotonin rescues insulin secretion in these mice. Interestingly, serotonin mainly acts not as an intercellular signaling molecule via its traditional surface receptors but intracellularly via regulation of the activity of target proteins through covalent coupling of serotonin to them. This coupling, called serotonylation, activates specific small GTPases, which in turn promote glucose-mediated insulin secretion. Adding this receptor-independent signaling mechanism to the multifarious regulatory functions of serotonin, we hypothesize that protein serotonylation modulates physiological secretion processes in all serotonin-containing tissues.

Immune regulation by 4‐1BB and 4‐1BBL: complexities and challenges

The tumor necrosis factor receptor family member 4-1BB plays a key role in the survival of activated and memory CD8(+) T cells. Depending on the disease model, 4-1BB can participate at different stages and influence different aspects of the immune response, likely due to the differential expression of receptor and ligand relative to other costimulatory molecules. Studies comparing mild versus severe influenza infection of mice suggest that the immune system uses inducible receptors such as 4-1BB to prolong the immune response when pathogens take longer to clear. The expression of 4-1BB on diverse cell types, evidence for bidirectional as well as receptor-independent signaling by 4-1BBL, the unexpected hyperproliferation of 4-1BB-deficient T cells, and complex effects of agonistic anti-4-1BB therapy have revealed additional roles for the 4-1BB/4-1BBL receptor/ligand pair in the immune system. In this review, we discuss these diverse roles of 4-1BB and its ligand in the immune response, exploring possible mechanisms for the observed complexities and implications for therapeutic applications of 4-1BB/4-1BBL.

Modulation of Δ 9-tetrahydrocannabinol-induced MCF-7 breast cancer cell growth by cyclooxygenase and aromatase

Δ 9-Tetrahydrocannabinol (Δ 9-THC), a major constituent of marijuana, has been shown to stimulate the growth of MCF-7 breast cancer cells through cannabinoid receptor-independent signaling [Takeda, S., Yamaori, S., Motoya, E., Matsunaga, T., Kimura, T., Yamamoto, I., Watanabe, K., 2008. Δ 9-Tetrahydrocannabinol enhances MCF-7 cell proliferation via cannabinoid receptor-independent signaling. Toxicology 245, 141–146]. Although the growth of MCF-7 cells is known to be stimulated by 17β-estradiol (E 2), the interaction of Δ 9-THC and E 2 in MCF-7 cell growth is not fully clarified so far. In the present study, by using E 2-sensitive MCF-7 cells that have expressed cyclooxygenase-2 (COX-2) and cytochrome P450 19 (aromatase), we studied whether or not COX-2 and aromatase are involved in Δ 9-THC-mediated MCF-7 cell proliferation. It was shown that Δ 9-THC-induced MCF-7 cell growth was inhibited by COX-2 inhibitors and was stimulated by arachidonic acid (a COX substrate). However, the growth of MCF-7 cells induced by Δ 9-THC was not stimulated by PGE 2, and the expression of aromatase was not affected by COX-2 inhibitors, arachidonic acid, and PGE 2, suggesting that there is a disconnection between COX-2 (PGE 2) and aromatase in Δ 9-THC-mediated MCF-7 cell proliferation. On the other hand, Δ 9-THC-induced MCF-7 cell growth was elevated by two kinds of aromatase inhibitors. Taken together with the evidence that Δ 9-THC-induced MCF-7 cell proliferation was interfered with testosterone (an aromatase substrate) and exogenously provided E 2, it is suggested that (1) the growth stimulatory effects of Δ 9-THC are mediated by the product(s) of COX-2 except for PGE 2, (2) the action of Δ 9-THC is modulated by E 2, and (3) COX-2 and aromatase are individually engaged in the proliferation of MCF-7 cells induced by Δ 9-THC.

Comprehensive Dissection of PDGF-PDGFR Signaling Pathways in PDGFR Genetically Defined Cells

Despite the growing understanding of PDGF signaling, studies of PDGF function have encountered two major obstacles: the functional redundancy of PDGFRα and PDGFRβ in vitro and their distinct roles in vivo. Here we used wild-type mouse embryonic fibroblasts (MEF), MEF null for either PDGFRα, β, or both to dissect PDGF-PDGFR signaling pathways. These four PDGFR genetically defined cells provided us a platform to study the relative contributions of the pathways triggered by the two PDGF receptors. They were treated with PDGF-BB and analyzed for differential gene expression, in vitro proliferation and differential response to pharmacological effects. No genes were differentially expressed in the double null cells, suggesting minimal receptor-independent signaling. Protean differentiation and proliferation pathways are commonly regulated by PDGFRα, PDGFRβ and PDGFRα/β while each receptor is also responsible for regulating unique signaling pathways. Furthermore, some signaling is solely modulated through heterodimeric PDGFRα/β.

Protease‐activated receptor dependent and independent signaling by kallikreins 1 and 6 in CNS neuron and astroglial cell lines

While protease-activated receptors (PARs) are known to mediate signaling events in CNS, contributing both to normal function and pathogenesis, the endogenous activators of CNS PARs are poorly characterized. In this study, we test the hypothesis that kallikreins (KLKs) represent an important pool of endogenous activators of CNS PARs. Specifically, KLK1 and KLK6 were examined for their ability to evoke intracellular Ca(2+) flux in a PAR-dependent fashion in NSC34 neurons and Neu7 astrocytes. Both KLKs were also examined for their ability to activate mitogen-activated protein kinases (extracellular signal-regulated kinases, C-Jun N-terminal kinases, and p38) and protein kinase B (AKT) intracellular signaling cascades. Cumulatively, these studies show that KLK6, but not KLK1, signals through PARs. KLK6 evoked intracellular Ca(2+) flux was mediated by PAR1 in neurons and both PAR1 and PAR2 in astrocytes. Importantly, both KLK1 and KLK6 altered the activation state of mitogen-activated protein kinases and AKT, suggestive of important roles for each in CNS neuron and glial differentiation, and survival. The cellular specificity of CNS-KLK activity was underscored by observations that both proteases promoted AKT activation in astrocytes, but inhibited such signaling in neurons. PAR1 and bradykinin receptor inhibitors were used to demonstrate that KLK1-mediated activation of extracellular signal-regulated kinases in neurons occurred in a non-PAR, bradykinin 2 (B2) receptor-dependent fashion, while similar signaling by KLK6 was mediated by the combined activation of PAR1 and B2. Cumulatively results indicate KLK6, but not KLK1 is an activator of CNS PARs, and that both KLKs are poised to signal in a B2 receptor-dependent fashion to regulate multiple signal transduction pathways relevant to CNS physiologic function and dysfunction.