Graeme F. Nixon

Sphingolipids in inflammation: pathological implications and potential therapeutic targets

Sphingolipids are formed via the metabolism of sphingomyelin, a constituent of the plasma membrane, or by de novo synthesis. Enzymatic pathways result in the formation of several different lipid mediators, which are known to have important roles in many cellular processes, including proliferation, apoptosis and migration. Several studies now suggest that these sphingolipid mediators, including ceramide, ceramide 1‐phosphate and sphingosine 1‐phosphate (S1P), are likely to have an integral role in inflammation. This can involve, for example, activation of pro‐inflammatory transcription factors in different cell types and induction of cyclooxygenase‐2, leading to production of pro‐inflammatory prostaglandins. The mode of action of each sphingolipid is different. Increased ceramide production leads to the formation of ceramide‐rich areas of the membrane, which may assemble signalling complexes, whereas S1P acts via high‐affinity G‐protein‐coupled S1P receptors on the plasma membrane. Recent studies have demonstrated that in vitro effects of sphingolipids on inflammation can translate into in vivo models. This review will highlight the areas of research where sphingolipids are involved in inflammation and the mechanisms of action of each mediator. In addition, the therapeutic potential of drugs that alter sphingolipid actions will be examined with reference to disease states, such as asthma and inflammatory bowel disease, which involve important inflammatory components. A significant body of research now indicates that sphingolipids are intimately involved in the inflammatory process and recent studies have demonstrated that these lipids, together with associated enzymes and receptors, can provide effective drug targets for the treatment of pathological inflammation.

Comparison of Sphingosine 1-Phosphate--Induced Intracellular Signaling Pathways in Vascular Smooth Muscles. Differential Role in Vasoconstriction

Sphingosine 1-phosphate (S1P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S1P) family of 7 transmembrane G protein–coupled receptors. In cultured vascular smooth muscle (VSM) cells, S1P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S1P and EDG/S1P receptor expression were determined in rat VSM from cerebral artery and aorta. S1P induced constriction of cerebral artery, which was partly dependent on activation of p160ROCK (Rho-kinase). S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S1P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S1P gives rise to a global increase in [Ca2+]i, initially generated via Ca2+ release from the sarcoplasmic reticulum by an inositol 1,4,5-trisphosphate–dependent pathway. In aorta VSM, a small increase in [Ca2+]i was observed after stimulation at higher concentrations of S1P. S1P induced activation of p42/p44mapk in aorta and cerebral artery VSM. Subtype-specific S1P receptor antibodies revealed that the expression of S1P3/EDG-3 and S1P2/EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S1P1/EDG-1 receptor expression was similar in both types of VSM. Therefore, the ability of S1P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S1P receptor subtypes.

Localization of Ryanodine Receptors in Smooth Muscle

The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with anti-RyR antibodies showed a patchy network-like staining pattern throughout the cell cytoplasm, excluding nuclei, in aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanide-stained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti-RyR antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR-Ca2+-release channels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe analysis, which show a decrease in the Ca2+ content of both peripheral and internal SR in stimulated smooth muscles. The complex distribution of inositol 1,4,5-trisphosphate and ryanodine receptors (present study) is compatible with their proposed roles as agonist-induced Ca2+-release channels and origins of Ca2+ sparks, Ca2+ oscillations, and Ca2+ waves.

Tumour necrosis factor-α activates a calcium sensitization pathway in guinea-pig bronchial smooth muscle

1 The effects of tumour necrosis factor‐α (TNF) on guinea‐pig bronchial smooth muscle contractility were investigated. 2 The Ca2+‐activated contractile response of permeabilized bronchial smooth muscle strips was significantly increased after incubation with 1 μg ml−1 TNF for 45 min. This TNF‐induced effect was not due to a further increase in intracellular Ca2+. 3 The TNF‐induced Ca2+ sensitization was, at least partly, the result of an increase in myosin light chain20 phosphorylation. 4 The intracellular signalling pathway involved in this effect of TNF was further investigated. Sphingomyelinase, a potential mediator of TNF, had no effect on Ca2+ sensitivity of permeabilized bronchial smooth muscle. Also, p42/p44 mitogen‐activated protein kinase (p42/p44mapk), activated by TNF in some cell types, did not show an increased activation in bronchial smooth muscle after TNF treatment. 5 In conclusion, TNF may activate a novel signalling pathway in guinea‐pig bronchial smooth muscle leading to an increase in myosin light chain20 phosphorylation and a subsequent increase in Ca2+ sensitivity of the myofilaments. This pathway does not appear to involve sphingomyelinase‐liberated ceramides or activation of p42/p44mapk. Given the importance of TNF in asthma, this TNF‐induced Ca2+ sensitization of the myofilaments may represent a mechanism responsible for airway hyper‐responsiveness.

Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle.

In arterial myocytes the Ca(2+) mobilizing messenger NAADP evokes spatially restricted Ca(2+) bursts from a lysosome-related store that are subsequently amplified into global Ca(2+) waves by Ca(2+)-induced Ca(2+)-release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) alphalgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5mum of the nucleus) was approximately 4-fold greater than in the sub-plasmalemmal (within 1.5mum of the plasma membrane) and approximately 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was approximately 4- and approximately 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and approximately 2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with alphalgp120 labelling within the perinuclear region was approximately 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alphalgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30muM) abolished global Ca(2+) waves but not Ca(2+) bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca(2+) signalling by NAADP.

Spatial Compartmentalization of Tumor Necrosis Factor (TNF) Receptor 1-dependent Signaling Pathways in Human Airway Smooth Muscle Cells LIPID RAFTS ARE ESSENTIAL FOR TNF-α-MEDIATED ACTIVATION OF RhoA BUT DISPENSABLE FOR THE ACTIVATION OF THE NF-κB AND MAPK PATHWAYS

Tumor necrosis factor (TNF)-α-induced activation of RhoA, mediated by TNF receptor 1 (TNFR1), is a prerequisite step in a pathway that leads to increased 20-kDa light chain of myosin (MLC20) phosphorylation and airway smooth muscle contraction. In this study, we have investigated the proximal events in TNF-α-induced RhoA activation. TNFR1 is localized to both lipid raft and nonraft regions of the plasma membrane in primary human airway smooth muscle cells. TNF-α engagement of TNFR1 recruited the adaptor proteins TRADD, TRAF-2, and RIP into lipid rafts and activated RhoA, NF-κB, and MAPK pathways. Depletion of cholesterol from rafts with methyl-β-cyclodextrin caused a redistribution of TNFR1 to nonraft plasma membrane and prevented ligand-induced RhoA activation. By contrast, TNF-α-induced activation of NF-κB and MAPKs was unaffected by methyl-β-cyclodextrin indicating that, in airway smooth muscle cells, activation of these pathways occurred independently of lipid rafts. Targeted knockdown of caveolin-1 completely abrogated TNF-α-induced RhoA activation, identifying this raft-resident protein as a positive regulator of the activation process. The signaling adaptors TRADD and RIP were also found to be necessary for ligand-induced RhoA activation. Taken together, our results suggest that in airway smooth muscle cells, spatial compartmentalization of TNFR1 provides a mechanism for generating distinct signaling outcomes in response to ligand engagement and define a mechanistic role for lipid rafts and caveolin-1 in TNF-α-induced activation of RhoA.

ETA receptors are the primary mediators of myofilament calcium sensitization induced by ET-1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway

We have investigated the possibility that ET‐1 can induce an increase in myofilament calcium sensitivity in pulmonary artery smooth muscle. Arterial rings were permeabilized using α‐toxin (120u2003μgu2003ml−1), in the presence of A23187 (10u2003μM) to ‘knock out’ Ca2+ stores, and pre‐constricted with pCau20036.8 (buffered with 10u2003mM EGTA). In the presence of this fixed Ca2+ concentration, 1u2003μM ET‐1 induced a sustained, reversible constriction of 0.15u2003mN. Pulmonary arterial rings were freeze‐clamped at the peak of the induced constriction (time matched). Subsequent densitometric analysis revealed that ET‐1 (1u2003μM) increased the level of phosphorylated myosin light chains by 34% compared to an 11% increase in the presence of pCau20036.8 alone. In contrast to ET‐1, the selective ETB receptor agonist Sarafotoxin S6C (100u2003nM) failed to induce a significant constriction. The constriction induced by 1u2003μM ET‐1 was reversibly inhibited when the preparation was pre‐incubated (15u2003min) with the ETA receptor antagonist BQu2003123 (100u2003μM). The constriction measured 0.13u2003mN in the absence and 0.07u2003mN in the presence of 100u2003μM BQu2003123. In contrast, the constriction induced by 1u2003μM ET‐1 measured 0.19u2003mN in the absence and 0.175u2003mN following a 15u2003min pre‐incubation with the ETB antagonist BQu2003788 (100u2003μM). The constriction induced by 1u2003μM ET‐1 measured 0.14u2003mN in the presence and 0.13u2003mN following pre‐incubation with the tyrosine kinase inhibitor Tyrphostinu2003A23 (100u2003μM). We conclude that ET‐1 induced an increase in myofilament calcium sensitivity in rat pulmonary arteries via the activation of ETA receptors and by a mechanism(s) independent of tyrosine kinase.

Sphingolipids differentially regulate mitogen‐activated protein kinases and intracellular Ca2+ in vascular smooth muscle: effects on CREB activation

Related sphingolipids, sphingosine 1‐phosphate (S1P) and sphingosylphosphorylcholine (SPC), have important effects on vascular smooth muscle. The aim of this study was to investigate the intracellular pathways regulated by S1P and SPC in rat cerebral artery. In cerebral arteries, S1P increased extracellular signal‐regulated kinase (ERK)1/2 phosphorylation (5.2±1.4‐fold increase) but did not activate p38 mitogen‐activated protein kinase (p38MAPK) as assessed by immunoblotting. In contrast, SPC increased p38MAPK phosphorylation (3.0±0.3‐fold increase) but did not stimulate ERK1/2. This differential activation was confirmed by measuring activation of heat shock protein (HSP) 27, a known downstream target of p38MAPK. Only SPC, but not S1P, activated HSP27. In enzymatically dispersed cerebral artery myocytes, SPC increased [Ca2+]i in a concentration‐dependent manner (peak response at 10u2003μM: 0.4±0.02 ratio units) as determined using the Ca2+ indicator, Fura 2. In contrast to S1P, the SPC‐induced [Ca2+]i increase did not involve intracellular release but was due to Ca2+ influx via L‐type Ca2+ channels. Despite differences in signalling, both S1P and SPC phosphorylated the transcription factor cAMP response element‐binding protein (CREB). S1P‐induced CREB activation was dependent on ERK1/2 and Ca2+‐calmodulin‐dependent protein kinase (CaMK) activation. CREB activation by SPC required both p38MAPK and CaMK activation, but not ERK1/2. In conclusion, S1P and SPC activate distinct MAP kinase isoforms and increase [Ca2+]i via different mechanisms in rat cerebral artery. This does not affect the ability of S1P or SPC to activate CREB, although this occurs via different pathways.

Stimulation of stress-activated but not mitogen-activated protein kinases by tumour necrosis factor receptor subtypes in airway smooth muscle

The multifunctional cytokine tumour necrosis factor-alpha (TNF) displays many physiological effects in a variety of tissues, especially proliferative and cytotoxic actions in immunological cells. Recently, we uncovered an important new mechanism by which TNF can sensitise airway smooth muscle (ASM) to a fixed intracellular Ca2+ concentration which in vivo would produce a marked hypercontractility of the airways. Here, we report that both 50-60 kDa type I TNFR (TNFR1) and 70-80 kDa type II TNFR (TNFR2) receptor subtypes were expressed in ASM cells and selectively activated the stress kinases, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (p38 MAPK). However, TNF caused no activation of p42/p44 MAPK or cytosolic phospholipase A(2) activity. In contrast, TNF stimulation of the TNFR1, but not the TNFR2, elicited nuclear factor-kappaB transcription factor function, a species known to be important in mediation of certain inflammatory cellular responses. This is the first report of TNF receptor subtypes in ASM cells causing selective kinase activation, which may prove important in therapeutic strategies for treating immune airway disorders such as chronic obstructive pulmonary disease and asthma.

Sphingosine 1-phosphate induces CREB activation in rat cerebral artery via a protein kinase C-mediated inhibition of voltage-gated K+ channels.

Sphingosine 1-phosphate (S1P) is a potential mitogenic stimulus for vascular smooth muscle. S1P promotes an increase in the intracellular calcium concentration ([Ca(2+)](i)) in cerebral arteries, however S1P effects on regulation of gene expression are not known. Activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein (CREB), is associated with smooth muscle proliferation. The aim of this study was to examine the Ca(2+)-dependent mechanisms involved in S1P-induced CREB activation in cerebral artery. Western blotting and immunofluorescence with a phospho-CREB antibody were used to detect CREB activation in Sprague-Dawley rat cerebral arteries. Whole-cell patch clamp recording and single cell imaging of [Ca(2+)](i) were performed on freshly isolated cerebral artery myocytes. S1P increased activation of CREB in the nucleus of cerebral arteries. This activation was mediated by Ca(2+)/calmodulin-dependent protein kinase and was dependent on an increase in [Ca(2+)](i) via two mechanisms: (i) intracellular Ca(2+) release via an inositol 1,4,5-trisphosphate (InsP(3))-dependent pathway and (ii) Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Activation of the VDCC occurred through S1P-induced inhibition (approximately 50%) of the voltage-gated potassium (K(+)) current. This inhibition was via a protein kinase C-mediated pathway resulting in tyrosine phosphorylation of at least one isoform of the Kv channel (Kv 1.2). These results demonstrate that S1P can activate the transcription factor CREB through different Ca(2+)-dependent pathways including intracellular Ca(2+) release and inhibition of voltage-gated K(+) channels leading to Ca(2+) influx. Our findings suggest a potential role for S1P in regulation of gene expression in vascular smooth muscle.