Transforming growth factor-beta (TGF-beta) signaling plays a pivotal role in extracellular matrix deposition by stimulating collagen production and other extracellular matrix proteins and by inhibiting matrix degradation. The present study was undertaken to define the role of <em>sphingosine</em> kinase (SphK) in TGF-beta signaling. TGF-beta markedly up-regulated SphK<em>1</em> mRNA and protein amounts and caused a prolonged increase in SphK activity in dermal fibroblasts. Concomitantly, TGF-beta reduced <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> phosphatase activity. Consistent with the changes in enzyme activity, corresponding changes in sphingolipid levels were observed such that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) was increased (approximately 2-fold), whereas <em>sphingosine</em> and ceramide were reduced after 24 h of TGF-beta treatment. Given the relatively early induction of SphK gene expression in response to TGF-beta, we examined whether SphK<em>1</em> may be involved in the regulation of TGF-beta-inducible genes that exhibit compatible kinetics, e.g. tissue inhibitor of metalloproteinase-<em>1</em> (TIMP-<em>1</em>). We demonstrate that decreasing SphK<em>1</em> expression by small interfering RNA (siRNA) blocked TGF-beta-mediated up-regulation of TIMP-<em>1</em> protein suggesting that up-regulation of SphK<em>1</em> contributes to the induction of TIMP-<em>1</em> in response to TGF-beta. The role of SphK<em>1</em> as a positive regulator of TIMP-<em>1</em> gene expression was further corroborated by using ectopically expressed SphK<em>1</em> in the absence of TGF-beta. Adenovirally expressed SphK<em>1</em> led to a 2-fold increase of endogenous S<em>1</em>P and to increased TIMP-<em>1</em> mRNA and protein production. In addition, ectopic SphK<em>1</em> and TGF-beta cooperated in TIMP-<em>1</em> up-regulation. Mechanistically, experiments utilizing TIMP-<em>1</em> promoter constructs demonstrated that the action of SphK<em>1</em> on the TIMP-<em>1</em> promoter is through the AP<em>1</em>-response element, consistent with the SphK<em>1</em>-mediated up-regulation of phospho-c-Jun levels, a key component of AP<em>1</em>. Together, these experiments demonstrate that SphK/S<em>1</em>P are important components of the TGF-beta signaling pathway involved in up-regulation of the TIMP-<em>1</em> gene.

<em>Sphingosine</em> kinase (SPHK) is a key enzyme producing important messenger <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK<em>1</em> isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol <em>1</em>2-myristate <em>1</em>3-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol <em>1</em>2-myristate <em>1</em>3-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling.

The past three decades have witnessed remarkable advances in our ability to target specific elements of the immune and inflammatory response, fuelled by advances in both biotechnology and disease knowledge. As well as providing superior treatments for immune-mediated inflammatory diseases (IMIDs), such therapies also offer unrivalled opportunities to study the underlying immunopathological basis of these conditions.In this review, we explore recent approaches to the treatment of IMIDs and the insights to pathobiology that they provide. We review novel biologic agents targeting the T-helper <em>1</em>7 axis, including therapies directed towards interleukin (IL)-<em>1</em>7 (secukinumab, ixekizumab, bimekizumab), IL-<em>1</em>7R (brodalumab), IL-<em>1</em>2/23p40 (ustekinumab, briakinumab) and IL-23p<em>1</em>9 (guselkumab, tildrakizumab, brazikumab, risankizumab, mirikizumab). We also present an overview of biologics active against type I and II interferons, including sifalumumab, rontalizumab, anifrolumab and fontolizumab. Emerging strategies to interfere with cellular adhesion processes involved in lymphocyte recruitment are discussed, including both integrin blockade (natalizumab, vedolizumab, etrolizumab) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor inhibition (fingolimod, ozanimod). We summarise the development and recent application of Janus kinase (JAK) inhibitors in the treatment of IMIDs, including first-generation pan-JAK inhibitors (tofacitinib, baricitinib, ruxolitinib, peficitinib) and second-generation selective JAK inhibitors (decernotinib, filgotinib, upadacitinib). New biologics targeting B-cells (including ocrelizumab, veltuzumab, tabalumab and atacicept) and the development of novel strategies for regulatory T-cell modulation (including low-dose IL-2 therapy and Tregitopes) are also discussed. Finally, we explore recent biotechnological advances such as the development of bispecific antibodies (ABT-<em>1</em>22, COVA322), and their application to the treatment of IMIDs.

Decreased oxygen availability evokes adaptive responses, which are primarily under the gene regulatory control of hypoxia inducible factor-<em>1</em> (HIF-<em>1</em>). Hypoxic cores of a growing tumor cell mass use this signaling circuit to gain access to further blood and nutrient supply that guarantees their continuing growth. Interestingly, NO shares with hypoxia the ability to block prolyl-hydroxylase (PHD) activity, and thus the ability to stabilize hypoxia inducible factor <em>1</em> alpha (HIF-<em>1</em> alpha). Under these conditions NO mimics hypoxia, which might contribute to tumor development. Stimulating/triggering innate immune responses associated with macrophage activation often correlated with iNOS induction and massive NO release, which is known to kill NO-sensitive tumors. However, this safeguard mechanism will only be effective if all tumor cells are eliminated because apoptotic death of tumor cells implies mechanisms to stop macrophages from attacking the survivors. Apoptotic cells release factors, among others <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which reprogram macrophages. Macrophage reprogramming shifts responses from a M<em>1</em> and thus pro-inflammatory and killing phenotype, to a M2 phenotype, which is anti-inflammatory and pro-angiogenic. These polarized tumor associated macrophages (TAM) are actively contributing to tumor development. Apparently NO uses distinct signaling pathways that could serve as an explanation to understand how NO affects tumor development. Some of these pathways, especially the ability of NO to mimic hypoxia at the level of HIF-<em>1</em> alpha, as well as the role of macrophage polarization by apoptotic cells with accompanying changes in the iNOS versus arginase ratio and activities, will be discussed to better understand how NO affects tumor growth.

Lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are potent bioactive phospholipids with specific and multiple effects on blood cells and cells of the vessel wall. Released by activated platelets, LPA and S<em>1</em>P mediate physiological wound healing processes such as vascular repair. Evidence is accumulating that these lipid mediators can, however, under certain conditions become athero- and thrombogenic molecules that might aggravate cardiovascular disease. For example, LPA present in minimally modified LDL and within the intima of atherosclerotic lesions may play a role in the early phase of atherosclerosis by inducing barrier dysfunction and increased monocyte adhesion of the endothelium, as well as in the late phase by triggering platelet activation and intra-arterial thrombus formation upon rupture of the atherosclerotic plaque. Moreover, LPA and S<em>1</em>P, by stimulating the proliferation of fibroblasts and by enhancing the survival of inflammatory cells are likely to play a central role in the excessive fibroproliferative and inflammatory response to vascular injury that characterizes the progression of atherosclerosis. Furthermore, LPA can cause the phenotypic dedifferentiation of medial vascular smooth muscle cells, and S<em>1</em>P is able to stimulate the migration and proliferation of intimal vascular smooth muscle cells; both processes ultimately lead to the formation of the neointima. Most importantly, as LPA and S<em>1</em>P bind to and activate multiple G-protein receptors, it emerges that the beneficial or harmful action of LPA and S<em>1</em>P are critically dependent on the expression profile of their receptor subtypes and their coupling to different signal transduction pathways in the target cells. By targeting specific subtypes of LPA and S<em>1</em>P receptors in selective cells of the vascular wall and blood, new strategies for the prevention and therapy of cardiovascular diseases can be envisioned.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P), the initial product of Sph degradation by Sph kinase, was shown to be a strong inhibitor of cell motility and phagokinesis of B<em>1</em>6 melanoma and other types of cells at <em>1</em>0-<em>1</em>00 nM concentration. It also inhibited "chemoinvasion" of tumor cells through a thick layer of Matrigel on a filter membrane. Such inhibitory effects were produced minimally or not at all by Sph, N-methyl derivatives of Sph, or other related sphingolipids and phospholipids. Sph-<em>1</em>-P did not inhibit cell proliferation or protein kinase C (PKC) activity, in contrast to Sph and N-methyl-Sph, which inhibit PKC activity and cell growth in general. Radiolabeled [3H]Sph and [<em>1</em>4C]N-methyl-Sph were rapidly incorporated into B<em>1</em>6 melanoma cells. However, [<em>1</em>4C]N-methyl-Sph was not metabolically converted into other compounds, whereas [3H]Sph was efficiently converted within <em>1</em>0 min to Sph-<em>1</em>-P, followed by conversion to other sphingolipids and phospholipids. The inhibitory effect of Sph-<em>1</em>-P on cell motility and tumor cell invasiveness could be a specific phenomenon independent of PKC and other known transmembrane signaling mechanisms, based on an unknown mechanism. It may directly affect organizational assembly of actin filaments. Since exogenous Sph is rapidly converted into Sph-<em>1</em>-P, some reported effects of Sph may be ascribable to such conversion.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) has attracted much attention as a possible second messenger controlling cell proliferation and motility and as an intracellular Ca(2+)-releasing agent. Here, we present evidence that SPP activates a G protein-coupled receptor in the plasma membrane of various cells, leading to increase in cytoplasmic Ca2+ concentration ([Ca2+]i), inhibition of adenylyl cyclase, and opening of G protein-regulated potassium channels. In human enbryonic kidney (HEK) cells, SPP potently (EC50, 2 nM) and rapidly increased [Ca2+]i in a pertussis toxin-sensitive manner. Pertussis toxin-sensitive increase in [Ca2+]i was also observed with sphingosylphosphorylcholine (EC50, 460 nM), whereas other sphingolipids, including ceramide-<em>1</em>-<em>phosphate</em>, N-palmitoyl-<em>sphingosine</em>, psychosine, and D-erythro-<em>sphingosine</em> at micromolar concentrations did not or only marginally increased [Ca2+]i. Furthermore, SPP inhibited forskolin-stimulated cAMP accumulation in HEK cells and increased binding of guanosine 5'3-O-(thio) tri<em>phosphate</em> to HEK cell membranes. Rapid [Ca2+]i responses were also observed in human transitional bladder carcinoma (J82) cells, monkey COS-<em>1</em> cells, mouse NIH 3T3 cells, Chinese hamster ovary (CHO-K<em>1</em>) cells, and rat C6 glioma cells, whereas human HL-60 leukemia cells and human erythroleukemia cells failed to respond to SPP. In guinea pig atrial myocytes, SPP activated Gi protein-regulated inwardly rectifying potassium channels. Activation of these channels occurred strictly when SPP was applied at the extracellular face of atrial myocyte plasma membrane as measured in cell-attached and inside-out patch clamp current recordings. We conclude that SPP, in addition to its proposed direct action on intracellular Ca2+ stores, interacts with a high affinity Gi protein-coupled receptor in the plasma membrane of apparently many different cell types.

OBJECTIVE

To investigate the role of lysophospholipid growth factors in the regulation of aqueous humor outflow in the trabecular meshwork (TM).

METHODS

The expression profile of the endothelial differentiation gene (Edg) family of G-protein coupled receptors was determined by RT-PCR of human TM (HTM) cell-derived total RNA and by PCR amplification of HTM cell-derived and tissue-derived cDNA libraries. The effects of lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) on actin cytoskeleton and focal adhesions and on myosin light-chain (MLC) phosphorylation in HTM cells were evaluated by immunofluorescence microscopy and Western blot analysis, respectively. Activation of Rho GTPase in HTM cells was quantified by "pull-down" assays. Mobilization of intracellular calcium in HTM cells was determined using spectrofluorometric digital-imaging microscopy. The effects of LPA and S<em>1</em>P on aqueous humor outflow facility were evaluated by perfusion of enucleated porcine eyes.

RESULTS

Each of the receptor isoforms Edg<em>1</em>, -2, -3, and -4 was readily detectable in three of four HTM cell-derived libraries, whereas Edg2 was detectable in the HTM tissue library. LPA (20 microM) and S<em>1</em>P (<em>1</em> microM) stimulated actin stress fiber and focal adhesion formation, increased MLC phosphorylation, and induced marked activation of Rho GTPase in HTM cells. Both LPA (20 microM) and S<em>1</em>P (<em>1</em>0 microM) also stimulated increases in intracellular calcium concentration in HTM cells. LPA- and S<em>1</em>P-induced effects on MLC phosphorylation in HTM cells were markedly inhibited by pretreatment with the Rho kinase-specific inhibitor Y-27632 (5 microM). Perfusion of LPA (50 microM) and S<em>1</em>P (5 microM) in enucleated porcine eyes produced a significant decrease in aqueous humor outflow facility from baseline of 37% (n = 6) and 3<em>1</em>% (n = 5), respectively.

CONCLUSIONS

These studies demonstrate that LPA and S<em>1</em>P, the physiological agonists of Edg receptors, decrease outflow facility in perfused porcine eyes in association with increased MLC phosphorylation and Rho guanosine triphosphatase (GTPase) activation. These data provide evidence for a novel mechanism for negative regulation of outflow facility, which may contribute to overall physiological homeostasis of aqueous humor outflow facility.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and its receptor S<em>1</em>P<em>1</em> control T-cell egress from thymus and secondary lymphoid organs (SLOs). To further define the role of S<em>1</em>P<em>1</em> in lymphocyte trafficking, we performed adoptive transfer experiments and intravital microscopy (IVM) using both S<em>1</em>P<em>1</em>-/- lymphocytes and recipient wild-type (WT) mice treated with FTY720, an immunosuppressant that downmodulates S<em>1</em>P receptors. S<em>1</em>P<em>1</em> deficiency and FTY720 caused rapid disappearance of T cells from blood, prolonged retention in SLOs, and accumulation in bone marrow, but did not alter interstitial T-cell motility in peripheral lymph nodes (PLNs) as assessed by multiphoton IVM. However, S<em>1</em>P<em>1</em>-/- lymphocytes displayed reduced short-term homing to PLNs due to attenuated integrin-mediated firm arrest in high endothelial venules (HEVs). By contrast, S<em>1</em>P<em>1</em>-/- T cells homed normally to Peyer patches (PPs), whereas S<em>1</em>P<em>1</em>-/- B cells had a marked defect in homing to PPs and arrested poorly in PP HEVs. Therefore, S<em>1</em>P<em>1</em> not only controls lymphocyte egress from SLOs, but also facilitates in a tissue- and subset-specific fashion integrin activation during homing. Interestingly, FTY720 treatment enhanced accumulation of both S<em>1</em>P<em>1</em> sufficient and S<em>1</em>P<em>1</em>-/- T cells in PPs by enhancing integrin-mediated arrest in HEVs. Thus, FTY720 exerts unique effects on T-cell traffic in PPs that are independent of T-cell-expressed S<em>1</em>P<em>1</em>.

The clinical immunosuppressant FTY720 is a <em>sphingosine</em> analogue that, once phosphorylated by <em>sphingosine</em> kinase 2 (Sphk2), is an agonist of multiple receptor subtypes for <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Short exposures to FTY720 afford long term protection in lymphoproliferative and autoimmune disease models, presumably by inducing apoptosis in subsets of cells essential for pathogenesis. <em>Sphingosine</em> itself is pro-apoptotic, and apoptosis induced with FTY720 or <em>sphingosine</em> is thought to proceed independently of their phosphorylation. Following chemical mutagenesis of Jurkat cells we isolated mutants that are selectively resistant to FTY720 analogue AAL(R), as well as natural sphingolipid bases, including <em>sphingosine</em>. Cells lacking functional Sphk2 were resistant to apoptosis induced with AAL(R), indicating that apoptosis proceeds through AAL(R) phosphorylation. Phosphorylation of AAL(R) was also required for induction of lymphocyte apoptosis in mice, as apoptosis was not induced with the non-phosphorylatable chiral analogue, AAL(S). Apoptosis was induced in the spleen but not the thymus of mice administered <em>1</em> mg/kg AAL(R), correlating with levels of AAL(R)-<em>phosphate</em> (AFD(R)) in organ extracts. AFD(R) did not induce apoptosis when added to the cell culture medium, indicating that it induces apoptosis through an intracellular target. NBD-labeled AAL(R) localized to the endoplasmic reticulum, and AAL(R) treatment resulted in elevated cytosolic calcium, Bax redistribution from cytosol to mitochondrial and endoplasmic reticulum membranes, and caspase-independent mitochondrial permeabilization in Jurkat cells. We therefore describe an apoptotic pathway triggered by intracellular accumulation of sphingolipid base <em>phosphates</em> and suggest that sphingoid base substrates for Sphk2 acting intracellularly could be useful in the treatment of lymphoproliferative diseases.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a pleiotropic lipid mediator that has been shown to regulate cell growth, cell survival, cell invasion, vascular maturation, and angiogenesis, processes that are important for cancer progression. In this issue of Cancer Cell, Visentin et al. demonstrate that a monoclonal antibody that binds S<em>1</em>P with extremely high affinity and specificity significantly slows tumor progression and associated angiogenesis in several animal models of human cancer. Their results suggest that S<em>1</em>P not only affects tumor cells themselves, but also is permissive or required for the actions of angiogenic factors, and thus may be a bona fide cancer target.

BACKGROUND

NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca2+. We tested whether a cGMP-dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway.

RESULTS

The Ca2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca2+]i in depolarized RA (<em>1</em>20 mol/L K+) exposed to stepwise increases in Ca2+ex (0 to 3 mmol/L). Effects of <em>1</em>0 micromol/L sodium nitroprusside (SNP) on Ca2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (<em>1</em> micromol/L) and the MLCP inhibitor calyculin A (<em>1</em>20 nmol/L) and in presence of the RhoA-activating phospholipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P, <em>1</em>2 nmol/L). SNP-induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant-negative RhoA mutant (N<em>1</em>9RhoA). Constrictions elicited by increasing Ca2+ex were significantly attenuated by SNP, which, however, left associated increases in [Ca2+]i unaffected. This NO-induced attenuation was blocked by ODQ, calyculin A, and S<em>1</em>P. The S<em>1</em>P-induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N<em>1</em>9RhoA or Y27632 significantly augmented SNP-induced dilations.

CONCLUSIONS

NO dilates RA by activating the MLCP in a cGMP-dependent manner, thereby reducing the apparent Ca2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca2+-desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors.

Endothelial cell (EC) barrier dysfunction (i.e., increased vascular permeability) is observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that preserve vascular integrity have important clinical therapeutic implications. We examined the effects of methylnaltrexone (MNTX), a mu opioid receptor (mOP-R) antagonist, on human pulmonary EC barrier disruption produced by edemagenic agents including morphine, the endogenous mOP-R agonist DAMGO, thrombin, and LPS. Pretreatment of EC with MNTX (0.<em>1</em> muM, <em>1</em> h) or the uncharged mOP-R antagonist naloxone attenuated morphine- and DAMGO-induced barrier disruption in vitro. However, MNTX, but not naloxone, pretreatment of EC inhibited thrombin- and LPS-induced barrier disruption, indicating potential mOP-R-independent effects of MNTX. In addition, intravenously delivered MNTX attenuated LPS-induced vascular hyperpermeability in the murine lung. We next examined the mechanistic basis for this MNTX barrier protection and observed that silencing of mOP-R attenuated the morphine- and DAMGO-induced EC barrier disruption, but not the permeability response to either thrombin or LPS. Because activation of the <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor, S<em>1</em>P(3), is key to a number of barrier-disruptive responses, we examined the role of this receptor in the permeability response to mOP-R ligation. Morphine, DAMGO, thrombin, and LPS induced RhoA/ROCK-mediated threonine phosphorylation of S<em>1</em>P(3), which was blocked by MNTX, suggesting S<em>1</em>P(3) transactivation. In addition, silencing of S<em>1</em>P(3) receptor expression (siRNA) abolished the permeability response to each edemagenic agonist. These results indicate that MNTX provides barrier protection against edemagenic agonists via inhibition of S<em>1</em>P(3) receptor activation and represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.

S<em>1</em>P<em>1</em> (also known as EDG-<em>1</em>) is a G-protein coupled receptor for the bioactive lipid, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). Activation of S<em>1</em>P<em>1</em> receptor in endothelial cells induces diverse cellular effects, including cell proliferation, survival, migration and morphogenesis. Recent in vivo studies showed that the S<em>1</em>P<em>1</em> receptor is required in vascular maturation during development. While a number of studies reported a functional role of S<em>1</em>P<em>1</em> in vascular system and the presence of S<em>1</em>P<em>1</em> transcripts in various mouse organs, tissue distribution of S<em>1</em>P<em>1</em> has not been fully defined. In this study, we determined the expression pattern of S<em>1</em>P<em>1</em> by beta-galactosidase reporter gene expression, which is knocked into the S<em>1</em>P<em>1</em> locus. We show that S<em>1</em>P<em>1</em> is widely expressed in various cell types of adult mouse tissues, suggesting a regulatory role of this receptor in numerous physiological processes in both vascular and non-vascular tissues.

The bioactive lysophospholipids, primarily lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), are recent additions to the list of potent mediators of tissue repair and wound healing. In this review, we highlight the diverse actions of LPA and S<em>1</em>P on many types of cells involved in the wound healing process, with special emphasis on their regulation of fibroblasts. The effects of LPA and S<em>1</em>P are principally mediated via specific cell surface receptors. Important signaling pathways downstream of these receptors and the importance of TGFbeta and S<em>1</em>P cross-talk for wound healing are also discussed. Moreover, specific agonists and antagonists of the lysophospholipid receptors may be useful for the treatment of wounds and abnormal wound healing.

OBJECTIVE

Following injury, fibroblasts transform into myofibroblasts and produce extracellular matrix (ECM). Excess production of ECM associated with cardiac fibrosis severely inhibits cardiac function. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a bioactive lysophospholipid, regulates the function of numerous cell types. In this study, we determined the role of S<em>1</em>P in promoting pro-fibrotic actions of cardiac fibroblasts (CFs).

RESULTS

S<em>1</em>P-mediated effects on myofibroblast transformation, collagen production, and cross-talk with transforming growth factor-beta (TGF-beta) using mouse CF were examined. S<em>1</em>P increased alpha-smooth muscle actin (a myofibroblast marker) and collagen expression in a S<em>1</em>P2 receptor- and Rho kinase-dependent manner. TGF-beta increased sphingosine kinase <em>1</em> (SphK<em>1</em>; the enzyme responsible for S<em>1</em>P production) expression and activity. TGF-beta-stimulated collagen production was inhibited by SphK<em>1</em> or S<em>1</em>P2 siRNA, a SphK inhibitor, and an anti-S<em>1</em>P monoclonal antibody.

CONCLUSIONS

These findings suggest that TGF-beta-stimulated collagen production in CF involves 'inside-out' S<em>1</em>P signalling whereby S<em>1</em>P produced intracellularly by SphK<em>1</em> can be released and act in an autocrine/paracrine fashion to activate S<em>1</em>P2 and increase collagen production.

The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (<em>1</em>-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (<em>1</em>0-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT<em>1</em>. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) signaling via S<em>1</em>P<em>1</em> improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

We have identified the yeast <em>sphingosine</em> resistance gene (YSR2) of Saccharomyces cerevisiae as encoding a protein that specifically dephosphorylates dihydro<em>sphingosine</em> <em>1</em>-<em>phosphate</em> (DHS-<em>1</em>-P), and we refer to this protein as dihydro<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> phosphatase. Overexpression of YSR2 conferred <em>sphingosine</em> resistance to the dihydro<em>sphingosine</em>-<em>1</em>-P lyase-defective mutant (JS<em>1</em>6) of S. cerevisiae, which is hypersensitive to <em>sphingosine</em>. The ysr2Delta deletion mutant of S. cerevisiae accumulated DHS-<em>1</em>-P compared with its wild type strain upon labeling with D-erythro-[4, 5-3H]dihydro<em>sphingosine</em>, whereas overexpression of YSR2 increased dephosphorylation of DHS-<em>1</em>-P. An epitope-tagged fusion protein (YSR2-Flag) was partially purified and found to specifically dephosphorylate DHS-<em>1</em>-P to yield dihydro<em>sphingosine</em>. YSR2 failed to dephosphorylate ceramide <em>1</em>-<em>phosphate</em> or phosphatidic acid. Functionally, the mutant bearing the ysr2Delta deletion decreased labeling of sphingolipids and increased labeling of glycerolipids dramatically following in vivo labeling with D-erythro-[3H]dihydro<em>sphingosine</em>, but it slightly affected labeling of sphingolipids with inositol. Taken together, these results identify YSR2 as dihydro<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> phosphatase. They also raise the intriguing possibility that phosphorylation followed by dephosphorylation is required for incorporation of exogenous long chain sphingoid bases into sphingolipids.

It has long been known that sphingolipids, especially sphingomyelin, a principal component of myelin, are highly enriched in the central nervous system and are structural components of all eukaryotic cell membranes. In the last few years, substantial evidence has accumulated from studies of many types of cells demonstrating that in addition to their structural roles, their breakdown products form a new class of signaling molecules with potent and myriad regulatory effects on essentially every cell in the body. While the sphingolipid metabolites <em>sphingosine</em> and its precursor ceramide have been associated with cell growth arrest and apoptosis, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) enhances proliferation, differentiation, and cell survival as well as regulates many physiological and pathological processes. The relative levels of these three interconvertible sphingolipid metabolites, and thus cell fate, are strongly influenced by the activity of <em>sphingosine</em> kinases, of which there are two isoforms, designated SphK<em>1</em> and SphK2, the enzymes that phosphorylate <em>sphingosine</em> to produce S<em>1</em>P. Not much is yet known of the importance of S<em>1</em>P in the central nervous system. Therefore, this review is focused on current knowledge of regulation of SphK<em>1</em> and SphK2 on both transcriptional and post-translational levels and the functions of these isozymes and their product S<em>1</em>P and its receptors in the central nervous system.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) the product of <em>sphingosine</em> kinase (SK) action plays an important role in various pathological conditions like inflammation and cancer. In this study, we show that in the human breast cancer cell line MCF7, epidermal growth factor (EGF) stimulates SK-<em>1</em> activity in a biphasic manner with a first peak after <em>1</em>5 min and a second delayed activation occurring after <em>1</em> h up to <em>1</em>8 h and thereafter declining again. This delayed activation is accompanied by increased mRNA and protein expression of SK-<em>1</em>, but not SK-2. Mechanistically, the transcriptional upregulation is dependent on the classical mitogen-activated protein kinase, protein kinase C (PKC) and the phosphoinositide 3-kinase, since specific inhibitors of these enzymes all abolish the EGF-induced mRNA upregulation and activity of SK-<em>1</em>. Moreover, dexamethasone also suppressed EGF-induced SK-<em>1</em> mRNA expression and activity which is reversed by the glucocorticoid receptor antagonist RU486. To see whether EGF-induced upregulation of SK-<em>1</em> is of relevance for tumor progression, we investigated two hallmarks of carcinogenesis, i.e., cell proliferation and migration. Stimulation of cells with EGF leads to enhanced [(3)H]thymidine incorporation into DNA and also to stimulated migration in a modified Boyden chamber assay. When cells are depleted of SK-<em>1</em>, but not SK-2, by siRNA transfection or by dexamethasone treatment, EGF-induced proliferation and migration are drastically reduced. In summary, these data show that EGF causes an acute stimulation of SK-<em>1</em> activity and, moreover, triggers a delayed SK activation which is due to increased gene transcription and de novo synthesis of SK-<em>1</em>, which in turn directs cells towards growth and increased motility. Thus, the <em>sphingosine</em> kinase-<em>1</em> may represent a novel attractive target for cancer therapy.

We used a simple commercial magnetic immunobead method for the preparation of acutely isolated microglial cells from postnatal days <em>1</em>-3 rat brain. With the exception of a <em>1</em>5 min enzyme incubation, all stages are carried out at 4 degrees C, minimizing the opportunity for changes in gene expression during the isolation to be reflected in changes in accumulated mRNA. The composition of the isolated cells was compared with that of microglial cultures prepared by conventional tissue culture methods, and the purity of microglia was comparable between the two preparations. RT-PCR analysis of several genes related to inflammatory products indicated that the acutely prepared cells were in a less activated condition than the conventionally tissue cultured cells. We examined the pattern of expression of receptors for lysophosphatidic acid (lpa) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) using quantitative real-time PCR (TaqMan PCR) techniques. mRNA for LPA<em>1</em>, S<em>1</em>P<em>1</em>, S<em>1</em>P2, S<em>1</em>P3 and S<em>1</em>P5 was detected in these preparations, but the levels of the different receptor mRNAs varied according to the state of activation of the cells. mRNA for LPA3 was only detected significantly in cultured cell after lipopolysaccharide (LPS) stimulation, being almost absent in cultured microglia and undetectable in the acutely isolated preparations. The levels of mRNA of LPA<em>1</em> and S<em>1</em>P receptors was reduced by overnight exposure to S<em>1</em>P, while the same treatment significantly up-regulated the level of LPA3 mRNA.

Interactions between the Eph receptor tyrosine kinase and ephrin ligands transduce short-range signals regulating axon pathfinding, development of the cardiovascular system, as well as migration and spreading of neuronal and non-neuronal cells. Some of these effects are believed to be mediated by alterations in actin dynamics. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. EphrinA<em>1</em> is proposed to contribute to angiogenesis as it is strongly expressed at sites of neovascularization. Moreover, angiogenic factors induce the expression of ephrinA<em>1</em> in endothelial cells. In this study, using rat vascular smooth muscle cells (VSMCs), we investigated the contribution of the small Rho GTPases in ephrinA<em>1</em>-induced integrin inactivation. EphrinA<em>1</em> did not significantly affect early adhesion of VSMCs on purified laminin or fibronectin, but strongly impaired cell spreading. The Rho kinase inhibitor Y-27632 partly reversed the ephrinA<em>1</em> effect, suggesting involvement of Rho in this model. However, inhibition of RhoA synthesis with short interfering (si)RNA had a modest effect, suggesting that RhoA plays a limited role in ephrinA<em>1</em>-mediated inhibition of spreading in VSMCs. The ephrinA<em>1</em>-mediated morphological alterations correlated with inhibition of Rac<em>1</em> and p2<em>1</em>-activated kinase <em>1</em> (PAK<em>1</em>) activity, and were antagonized by the expression of a constitutively active Rac mutant. Moreover, repression of Rac<em>1</em> synthesis with siRNA amplifies the ephrinA<em>1</em>-induced inhibition of spreading. Finally, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lipid mediator known to inhibit Rac activation in VSMCs amplifies the ephrinA<em>1</em> effect. In conclusion, our results emphasize the role of the Rac/PAK pathway in ephrinA<em>1</em>-mediated inhibition of spreading. In this way, ephrinA<em>1</em>, alone or in synergy with S<em>1</em>P, can participate in blood vessel destabilization, a prerequisite for angiogenesis.

OBJECTIVE

Although the anti-EGF receptor (EGFR) monoclonal antibody cetuximab is an effective strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations in the "sphingolipid rheostat"-the balance between the proapoptotic molecule ceramide and the mitogenic factor <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P)-due to <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) overactivation have been involved in resistance to anticancer-targeted agents. Moreover, cross-talks between SphK<em>1</em> and EGFR-dependent signaling pathways have been described.

METHODS

We investigated SphK<em>1</em> contribution to cetuximab resistance in colorectal cancer, in preclinical in vitro/in vivo models, and in tumor specimens from patients.

RESULTS

SphK<em>1</em> was found overexpressed and overactivated in colorectal cancer cells with intrinsic or acquired resistance to cetuximab. SphK<em>1</em> contribution to resistance was supported by the demonstration that SphK<em>1</em> inhibition by N,N-dimethyl-<em>sphingosine</em> or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK<em>1</em> overexpression in sensitive cells confers resistance to these agents. Moreover, treatment of resistant cells with fingolimod (FTY720), a S<em>1</em>P receptor (S<em>1</em>PR) antagonist, resulted in resensitization to cetuximab both in vitro and in vivo, with inhibition of tumor growth, interference with signal transduction, induction of cancer cells apoptosis, and prolongation of mice survival. Finally, a correlation between SphK<em>1</em> expression and cetuximab response was found in colorectal cancer patients.

The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (<em>1</em>8:<em>1</em>Δ9) were nontoxic, whereas palmitoleate (<em>1</em>6:<em>1</em>Δ9) was a potent growth inhibitor. Cells treated with <em>1</em>6:<em>1</em>Δ9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthesis, and the release of solutes and low-molecular-weight proteins into the medium. Other cytotoxic lipids, such as glycerol ethers, <em>sphingosine</em>, and acyl-amines blocked growth by the same mechanisms. Nontoxic <em>1</em>8:<em>1</em>Δ9 was used for phospholipid synthesis, whereas toxic <em>1</em>6:<em>1</em>Δ9 was not and required elongation to <em>1</em>8:<em>1</em>Δ<em>1</em><em>1</em> prior to incorporation. However, blocking fatty acid metabolism using inhibitors to prevent acyl-acyl carrier protein formation or glycerol-<em>phosphate</em> acyltransferase activity did not increase the toxicity of <em>1</em>8:<em>1</em>Δ9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic <em>1</em>8:<em>1</em>Δ9 was as toxic as <em>1</em>6:<em>1</em>Δ9 in a strain lacking wall teichoic acids and led to growth arrest and enhanced release of intracellular contents. Thus, wall teichoic acids contribute to the structure-specific antimicrobial effects of unsaturated fatty acids. The ability of poorly metabolized <em>1</em>6:<em>1</em> isomers to penetrate the cell wall defenses is a weakness that has been exploited by the innate immune system to combat S. aureus.