Regulation of cell growth,survival and migration by ceramide 1-phosphate - implications in lung cancer progression and inflammation

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid that is implicated in the regulation of vital cellular functions and plays key roles in a number of inflammation-associated pathologies. C1P was first described as mitogenic for fibroblasts and macrophages and was later found to promote cell survival in different cell types. The mechanisms involved in the mitogenic actions of C1P include activation of MEK/ERK1-2, PI3K/Akt/mTOR, or PKC-α, whereas promotion of cell survival required a substantial reduction of ceramide levels through inhibition of serine palmitoyl transferase or sphingomyelinase activities. C1P and ceramide kinase (CerK), the enzyme responsible for its biosynthesis in mammalian cells, play key roles in tumor promotion and dissemination. CerK-derived C1P can be secreted to the extracellular milieu by different cell types and is also present in extracellular vesicles. In this context, whilst cell proliferation is regulated by intracellularly generated C1P, stimulation of cell migration/invasion requires the intervention of exogenous C1P. Regarding inflammation, C1P was first described as pro-inflammatory in a variety of cell types. However, cigarette smoke- or lipopolysaccharide-induced lung inflammation in mouse or human cells was overcome by pretreatment with natural or synthetic C1P analogs. Both acute and chronic lung inflammation, and the development of lung emphysema were substantially reduced by exogenous C1P applications, pointing to an anti-inflammatory action of C1P in the lungs. The molecular mechanisms involved in the regulation of cell growth, survival and migration with especial emphasis in the control of lung cancer biology are discussed.     

Control of inflammatory responses by ceramide,sphingosine 1-phosphate and ceramide 1-phosphate

Inflammation is a network of complex processes involving a variety of metabolic and signaling pathways aiming at healing and repairing damage tissue, or fighting infection. However, inflammation can be detrimental when it becomes out of control. Inflammatory mediators involve cytokines, bioactive lipids and lipid-derived metabolites. In particular, the simple sphingolipids ceramides, sphingosine 1-phosphate, and ceramide 1-phosphate have been widely implicated in inflammation. However, although ceramide 1-phosphate was first described as pro-inflammatory, recent studies show that it has anti-inflammatory properties when produced in specific cell types or tissues. The biological functions of ceramides and sphingosine 1-phosphate have been extensively studied. These sphingolipids have opposing effects with ceramides being potent inducers of cell cycle arrest and apoptosis, and sphingosine 1-phosphate promoting cell growth and survival. However, the biological actions of ceramide 1-phosphate have only been partially described. Ceramide 1-phosphate is mitogenic and anti-apoptotic, and more recently, it has been demonstrated to be key regulator of cell migration. Both sphingosine 1-phosphate and ceramide 1-phosphate are also implicated in tumor growth and dissemination. The present review highlights new aspects on the control of inflammation and cell migration by simple sphingolipids, with special emphasis to the role played by ceramide 1-phosphate in controlling these actions.     

A Novel Anti-Inflammatory and Pro-Resolving Role for Resolvin D1 in Acute Cigarette Smoke-Induced Lung Inflammation

Introduction

Cigarette smoke is a profound pro-inflammatory stimulus that contributes to acute lung injuries and to chronic lung disease including COPD (emphysema and chronic bronchitis). Until recently, it was assumed that resolution of inflammation was a passive process that occurred once the inflammatory stimulus was removed. It is now recognized that resolution of inflammation is a bioactive process, mediated by specialized lipid mediators, and that normal homeostasis is maintained by a balance between pro-inflammatory and pro-resolving pathways. These novel small lipid mediators, including the resolvins, protectins and maresins, are bioactive products mainly derived from dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFA). We hypothesize that resolvin D1 (RvD1) has potent anti-inflammatory and pro-resolving effects in a model of cigarette smoke-induced lung inflammation.

Methods

Primary human lung fibroblasts, small airway epithelial cells and blood monocytes were treated with IL-1β or cigarette smoke extract in combination with RvD1 in vitro, production of pro-inflammatory mediators was measured. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effects on lung inflammation and lung macrophage populations were assessed.

Results

RvD1 suppressed production of pro-inflammatory mediators by primary human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure.

Conclusions

RvD1 has potent anti-inflammatory and pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic approach to resolve lung injury caused by smoke and pulmonary toxicants.     

Relevance and potential of sphingosine-1-phosphate in vascular inflammatory disease

The typical pathological feature of atherosclerosis is inflammation. In the last years, it has become evident that inhibition of inflammation is one important therapeutic option in atherosclerosis. Recently, sphingolipid sphingosine-1-phosphate (S1P) was identified as a crucial molecule with potent anti-inflammatory properties. Indeed, S1P activates various G protein-coupled receptors, namely S1P1-S1P5. In the vasculature, mainly S1P1-3 receptors are present. FTY720, after phosphorylation to FTY720-P, is an orally active S1P mimetic. FTY720 has been developed for therapy in the field of autoimmune diseases and organ transplantation. In analogy to S1P, FTY720 shows potent anti-inflammatory effects and several groups have tested the in vivo effects of FTY720 on the progression of inflammatory vascular diseases. They could show that S1P receptor activation might lead to a partial inhibition of the progression of atherosclerotic lesions. S1P receptor activation therefore might be a concept for anti-inflammatory drug treatment. However, it is not clear how S1P and FTY720 exactly act on vascular inflammation. This review article gives a brief overview over the known actions of S1P in vascular inflammatory disease.     

Ceramide kinase regulates acute wound healing by suppressing 5-oxo-ETE biosynthesis and signaling via its receptor OXER1

The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group IVA cytosolic phospholipase A₂, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized. Herein, we determined that decreasing C1P activity via inhibitors or genetic ablation of the anabolic enzyme ceramide kinase (CERK) significantly enhanced wound healing phenotypes. Importantly, postwounding inhibition of CERK enhanced the closure rate of acute wounds, improved the quality of healing, and increased fibroblast migration via a “class switch” in the eicosanoid profile. This switch reduced pro-inflammatory prostaglandins (e.g., prostaglandin E2) and increased levels of 5-hydroxyeicosatetraenoic acid and the downstream metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE). Moreover, dermal fibroblasts from mice with genetically ablated CERK showed enhanced wound healing markers, while blockage of the murine 5-oxo-ETE receptor (oxoeicosanoid receptor 1) inhibited the enhanced migration phenotype of these cell models. Together, these studies reinforce the vital roles eicosanoids play in the wound healing process and demonstrate a novel role for CERK-derived C1P as a negative regulator of 5-oxo-ETE biosynthesis and the activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate.     

Liver: the formation and actions of aspirin-triggered lipoxins

Eicosanoids play a key role in the initiation, progression and resolution of the inflammatory response. Although most current anti-inflammatory strategies are focused on the pharmacological inhibition of pro-inflammatory eicosanoids, such as prostaglandins and leukotrienes, mounting evidence indicates the existence of potent endogenous eicosanoids able to control inflammation and orchestrate its resolution. The first eicosanoids recognized as anti-inflammatory compounds generated by our own organism were the lipoxins (LXs). More recently, a new series of carbon-15 epimers of LXs, with anti-inflammatory properties similar to those of native LXs, was identified during aspirin treatment. Since their formation is specific to this venerable non-steroidal anti-inflammatory drug, the term aspirin-triggered LXs (ATLs) was coined for these compounds. This chapter deals with the biosynthesis of LXs and ATLs in the liver, the largest solid organ/gland in the body, and discusses the most relevant actions of these lipid mediators in the context of liver inflammation and injury.     

TLR2/1 and sphingosine 1-phosphate modulate inflammation,myofibroblast differentiation and cell migration in fibroblasts

Dermal fibroblasts are important regulators of inflammatory and immune responses in the skin. The aim of the present study was to elucidate the interaction between two key players in inflammation, Toll-like receptors (TLRs) and sphingosine 1-phosphate (S1P), in normal human fibroblasts in the context of inflammation, fibrosis and cell migration. We demonstrate that TLR2 ligation strongly enhances the production of the pro-inflammatory cytokines IL-6 and IL-8. S1P significantly induces pro-inflammatory cytokines time- and concentration-dependently via S1P receptor (S1PR)2 and S1PR3. The TLR2/1 agonist Pam₃CSK₄ and S1P (> 1 μM) or TGF-β markedly upregulate IL-6 and IL-8 secretion. Pam₃CSK₄ and S1P alone promote myofibroblast differentiation as assessed by significant increases of α-smooth muscle actin and collagen I expression. Importantly, costimulation with S1P (> 1 μM) induces differentiation into myofibroblasts. In contrast, Pam₃CSK₄ and low S1P concentrations (< 1 μM) accelerate cell migration. These results suggest that TLR2/1 signaling and S1P cooperate in pro-inflammatory cytokine production and myofibroblast differentiation and promote cell migration of skin fibroblasts in a S1P-concentration dependent manner. Our findings provide significant insights into how infectious stimuli or danger signals and sphingolipids contribute to dermal inflammation which may be relevant for skin tissue repair after injury or disease.     

Targeting sphingosine-1-phosphate signaling for cancer therapy

Sphingosine-1-phosphate(S1P) is a potent pleotropic bioactive lipid mediator involved in immune cell trafficking, cell survival,cell proliferation, cell migration, angiogenesis and many other cellular processes. S1 P either activates S1 P receptors(S1PR1-5) through "inside-out signaling" or acts directly on intracellular targets to regulate various cellular processes. In the past two decades, much progress has been made in exploring S1 P signaling and its pathogenic roles in diseases as well as in developing modulators of S1 P signaling, including S1 P agonists, S1 P antagonists and sphingosine kinase(SphK) inhibitors.Ceramide and S1 P have been defined as reciprocal regulators of cell fate, and S1 P signaling has been shown to be crucial for the pathogenesis of various diseases, including autoimmune diseases, inflammation and cancer; therefore, targeting S1 P signaling may curtail the process of pathogenesis and serve as a potential therapeutic target for the treatment of these diseases. In this review, we describe recent advances in our understanding of S1 P signaling in cancer development(particularly in inflammationassociated cancer) as well as in innate and adaptive immunity, and we also discuss modulators of S1 P signaling in cancer treatment.     

Anti-inflammatory effect of the ceramide mixture extracted from genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The inflammatory response is an indispensable bodily reaction, but excessive inflammation is known to result in diseases such as atopic disease, bronchitis, rheumatoid arthritis, and inflammatory bowel disease. Ceramide is the basic structure of sphingolipids and ceramides have been industrially used in functional cosmetics as anti-aging agents, as well as for moisturizing skin and calming skin irritation. It also has been recently used in medicinal fields as an anti-inflammatory as well as for atopic and skin wound healing, and for skin barrier restoration. In this study, we used genetically modified Saccharomyces cerevisiae to produce ceramides. Ceramide mixture was produced by gene manipulation that amplifies the original yeast gene. To investigate their anti-inflammatory effects, nitric oxide (NO) concentrations in cell culture supernatant were measured by using the Griess reaction and the expression levels of pro-inflammatory markers, cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α), were determined by using qRT-PCR. When cells were treated with the ceramide mixture, cell viability was not reduced, but NO production was inhibited. In addition, expressions of COX-2 and TNF-α were inhibited. Based on these results, we conclude that ceramide biosynthesized from recombinant yeast can effectively reduce the expression of inflammatory enzymes and cytokines. We expected that ceramides biosynthesized in genetically modified yeast is a novel preventive or therapeutic agent for inflammatory diseases without the risk of foreign gene introduction.     

Downregulation of leukotriene biosynthesis by thymoquinone attenuates airway inflammation in a mouse model of allergic asthma

Chronic airway inflammation is a key feature of bronchial asthma. Leukotrienes are potent inflammatory mediators that play a role in the pathophysiology of asthma, and their levels are elevated in the airways in response to allergen challenge. We examined the anti-inflammatory effect of thymoquinone (TQ), the active principle in the volatile oil of Nigella sativa seeds, on leukotriene (LT) biosynthesis in a mouse model of allergic asthma. Mice sensitized and challenged with ovalbumin (OVA) antigen had an increased amounts of leukotriene B4 and C4, Th2 cytokines, and eosinophils in bronchoalveolar lavage (BAL) fluid. In addition, there was also a marked increase in lung tissue eosinophilia and goblet cell numbers. Administration of TQ before OVA challenge inhibited 5-lipoxygenase, the main enzyme in leukotriene biosynthesis, expression by lung cells and significantly reduced the levels of LTB4 and LTC4. This was accompanied by a marked decrease in Th2 cytokines and BAL fluid and lung tissue eosinophilia, all of which are characteristics of airway inflammation. These results demonstrate the anti-inflammatory effect of TQ in experimental asthma.     

Sphingosine kinase and sphingosine 1-phosphate in asthma

Sphingolipids are amphiphatic molecules ubiquitously expressed in all eukaryotic cell membranes. Initially characterized as structural components of cell membranes, sphingolipids have emerged as sources of important signalling molecules over the past decade. Sphingolipid metabolites, such as ceramide and S1P (sphingosine 1-phosphate), have been demonstrated to have roles as potent bioactive messengers involved in cell differentiation, proliferation, apoptosis, migration and angiogenesis. The importance of SphK (sphingosine kinase) and S1P in inflammation has been demonstrated extensively. The prevalence of asthma is increasing in many developed nations. Consequently, there is an urgent need for the development of new agents for the treatment of asthma, especially for patients who respond poorly to conventional therapy. Recent studies have demonstrated the important role of SphK and S1P in the development of asthma by regulating pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets in the treatment of asthma and are described in the present review.     

P1 receptors and cytokine secretion

Evidence has accumulated in the last three decades to suggest tissue protection and regeneration by adenosine in multiple different cell types. Adenosine produced in hypoxic or inflamed environments reduces tissue injury and promotes repair by receptor-mediated mechanisms. Among other actions, regulation of cytokine production and secretion by immune cells, astrocytes and microglia (the brain immunocytes) has emerged as a main mechanism at the basis of adenosine effects in diseases characterized by a marked inflammatory component. Many recent studies have highlighted that signalling through A₁ and A_2A adenosine receptors can powerfully prevent the release of pro-inflammatory cytokines, thus inhibiting inflammation and reperfusion injury. However, the activation of adenosine receptors is not invariably protective of tissues, as signalling through the A_2B adenosine receptor has been linked to pro-inflammatory actions which are, at least in part, mediated by increased release of pro-inflammatory cytokines from epithelial cells, astrocytes and fibroblasts. Here, we discuss the multiple actions of P1 receptors on cytokine secretion, by analyzing, in particular, the role of the various adenosine receptor subtypes, the complex reciprocal interplay between the adenosine and the cytokine systems, their pathophysiological significance and the potential of adenosine receptor ligands as new anti-inflammatory agents.     

Inhibitory effects of ceramide kinase on Rac1 activation,lamellipodium formation,cell migration,and metastasis of A549 lung cancer cells

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), a bioactive sphingolipid. Since the mechanisms responsible for regulating the proliferation and migration/metastasis of cancer cells by the CerK/C1P pathway remain unclear, we conducted the present study. The knockdown of CerK in A549 lung and MCF-7 breast cancer cells (shCerK cells) increased the formation of lamellipodia, which are membrane protrusions coupled with cell migration. Mouse embryonic fibroblasts prepared from CerK-null mice also showed an enhanced formation of lamellipodia. The overexpression of CerK inhibited lamellipodium formation in A549 cells. The knockdown of CerK increased the number of cells having lamellipodia with Rac1 and the levels of active Rac1-GTP form, whereas the overexpression of CerK decreased them. CerK was located in lamellipodia after the epidermal growth factor treatment, indicating that CerK functioned there to inhibit Rac1. The migration of A549 cells was negatively regulated by CerK. An intravenous injection of A549-shCerK cells into nude mice resulted in markedly stronger metastatic responses in the lungs than an injection of control cells. The in vitro growth of A549 cells and in vivo expansion after the injection into mouse flanks were not affected by the CerK knockdown. These results suggest that the activation of CerK/C1P pathway has inhibitory roles on lamellipodium formation, migration, and metastasis of A549 lung cancer cells.     

Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid Correlate with Clinical Measures of Disease

In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, and inflammation. Here we explored complement inflammatory effectors in CF lung fluid. In this study soluble fractions (sols) from sputum samples of 15 CF patients were assayed for complement effectors and analyzed with clinical measurements. The pro-inflammatory peptide C5a was increased 4.8-fold (P = 0.04) in CF sols compared with controls. Incubation of CF sols with P. aeruginosa or S. aureus increased C5a concentration 2.3-fold (P = 0.02). A peptide inhibitor of complement C1 (PIC1) completely blocked the increase in C5a concentration from P. aeruginosa in CF sol in vitro (P = 0.001). C5a concentration in CF sol correlated inversely with body mass index (BMI) percentile in children (r = -0.77, P = 0.04). C3a, which has anti-inflammatory effects, correlated positively with FEV1% predicted (r_s = 0.63, P = 0.02). These results suggest that complement effectors may significantly impact inflammation in CF lung fluid.     

A Short Peptide That Mimics the Binding Domain of TGF-β1 Presents Potent Anti-Inflammatory Activity

The transforming growth factor beta 1 (TGF-β1) is a pleiotropic cytokine with multiple roles in development, wound healing, and immune regulation. TGF-β1-mediated immune dysfunction may lead to pathological conditions, such as inflammation. Chronic inflammatory process is characterized by a continuous release of pro-inflammatory cytokines, and the inhibition or the blockage of these cytokines signaling pathways are considered a target treatment. In this context, despite the high numbers of TGF-β-targeted pathways, the inducible regulatory T cells (iTreg) to control inflammation seems to be a promising approach. Our aim was to develop novel peptides through phage display (PhD) technology that could mimic TGF-β1 function with higher potency. Specific mimetic peptides were obtained through a PhD subtraction strategy from whole cell binding using TGF-β1 recombinant as a competitor during elution step. We have selected a peptide that seems to play an important role on cellular differentiation and modulation of TNF-α and IL-10 cytokines. The synthetic pm26TGF-β1 peptide tested in PBMC significantly down-modulated TNF-α and up-regulated IL-10 responses, leading to regulatory T cells (Treg) phenotype differentiation. Furthermore, the synthetic peptide was able to decrease leukocytes rolling in BALB/C mice and neutrophils migration during inflammatory process in C57BL/6 mice. These data suggest that this peptide may be useful for the treatment of inflammatory diseases, especially because it displays potent anti-inflammatory properties and do not exhibit neutrophils’ chemoattraction.     

Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution

Lipoxins (LXs) or the lipoxygenase interaction products are generated from arachidonic acid via sequential actions of lipoxygenases and subsequent reactions to give specific trihydroxytetraene-containing eicosanoids. These unique structures are formed during cell-cell interactions and appear to act at both temporal and spatially distinct sites from other eicosanoids produced during the course of inflammatory responses and to stimulate natural resolution. Lipoxin A4 (LXA4) and lipoxin B4 (LXB4) are positional isomers that each possesses potent cellular and in vivo actions. These LX structures are conserved across species. The results of numerous studies reviewed in this work now confirm that they are the first recognized eicosanoid chemical mediators that display both potent anti-inflammatory and pro-resolving actions in vivo in disease models that include rabbit, rat, and mouse systems. LXs act at specific GPCRs as agonists to regulate cellular responses of interest in inflammation and resolution. Aspirin has a direct impact in the LX circuit by triggering the biosynthesis of endogenous epimers of LX, termed the aspirin-triggered 15-epi-LX, that share the potent anti-inflammatory actions of LX. Stable analogs of LXA4, LXB4, and aspirin-triggered lipoxin were prepared, and several of these display potent actions in vitro and in vivo. The results reviewed herein implicate a role of LX and their analogs in many common human diseases including airway inflammation, asthma, arthritis, cardiovascular disorders, gastrointestinal disease, periodontal disease, kidney diseases and graft-vs.-host disease, as well as others where uncontrolled inflammation plays a key role in disease pathogenesis. Hence, the LX pathways and mechanisms reviewed to date in this work provide a basis for new approaches to treatment of many common human diseases that involve inflammation.