sphingosine-1-phosphate and Hyperplasia

The type II transmembrane serine protease Matriptase 1 (ST14) is commonly known as an oncogene, yet it also plays an understudied role in suppressing carcinogenesis. This double face is evident in the embryonic epidermis of zebrafish loss-of-function mutants in the cognate Matriptase inhibitor Hai1a (Spint1a). Mutant embryos display epidermal hyperplasia, but also apical cell extrusions, during which extruding outer keratinocytes carry out an entosis-like engulfment and entrainment of underlying basal cells, constituting a tumor-suppressive effect. These counteracting Matriptase effects depend on EGFR and the newly identified mediator phospholipase D (PLD), which promotes both mTORC1-dependent cell proliferation and sphingosine-1-phosphate (S1P)-dependent entosis and apical cell extrusion. Accordingly, hypomorphic hai1a mutants heal spontaneously, while otherwise lethal hai1a amorphs are efficiently rescued upon cotreatment with PLD inhibitors and S1P. Together, our data elucidate the mechanisms underlying the double face of Matriptase function in vivo and reveal the potential use of combinatorial carcinoma treatments when such double-face mechanisms are involved.

Topics: Animals; Carcinogenesis; Cell Proliferation; Disease Models, Animal; Embryonic Development; Entosis; Epidermis; ErbB Receptors; Genes, Tumor Suppressor; Humans; Hyperplasia; Keratinocytes; Loss of Function Mutation; Lysophospholipids; Mechanistic Target of Rapamycin Complex 1; Phospholipase D; Proteinase Inhibitory Proteins, Secretory; Serine Endopeptidases; Sphingosine; Zebrafish

Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cell- and IgE-dependent murine model of allergic asthma has not yet been examined.. We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice.. Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge.. SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs.. S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.

Topics: Amino Alcohols; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokine CCL2; Female; Goblet Cells; Humans; Hyperplasia; Immunoglobulin E; Inflammation; Interferon-gamma; Interleukins; Lung; Lysophospholipids; Mast Cells; Methacholine Chloride; Mice; Mice, Inbred C57BL; NF-kappa B; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Necrosis Factor-alpha

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P(1)) and participates in many pathological conditions. We developed a novel type S1P(1)-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P(1) resulting in reduced signaling downstream of S1P(1), including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P(1)-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P(1) antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P(1) agonists on lymphocyte sequestration results from their functional antagonism.

Topics: Animals; Chemotaxis; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Dermatitis, Contact; Ear; Edema; Female; Guanosine 5'-O-(3-Thiotriphosphate); HEK293 Cells; Humans; Hyperplasia; Leukocytes; Lymphopenia; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Molecular Structure; Protein Binding; Rats; Rats, Inbred Lew; Receptors, Lysosphingolipid; Sphingosine; Sulfonamides; Triazoles

The objective of this study was to define a role for sphingosine-1-phosphate receptor 3 (S1PR3) in intimal hyperplasia.. A denudation model of the iliac-femoral artery in wild-type and S1PR3-null mice was used to define a role for S1PR3 in the arterial injury response because we found in humans and mice that expression of S1PR3 was higher in these arteries compared with carotid arteries. At 28 days after surgery, wild-type arteries formed significantly larger lesions than S1PR3-null arteries. Bromodeoxyuridine labeling experiments demonstrated that on injury, wild-type arteries exhibited higher medial as well as intimal proliferation than S1PR3-null arteries. Because S1PR3 expression in vitro was low, we expressed S1PR3 in S1PR3-null smooth muscle cells (SMCs) using retroviral-mediated gene transfer to study the effects of S1PR3 on cell functions and signaling. SMCs expressing S1PR3, but not vector-transfected controls, responded to sphingosine-1-phosphate stimulation with activation of Rac, Erk, and Akt. SMCs expressing S1PR3 also migrated more.. In humans and mice, S1PR3 expression was higher in iliac-femoral arteries compared with carotid arteries. S1PR3 promoted neointimal hyperplasia on denudation of iliac-femoral arteries in mice, likely by stimulating cell migration and proliferation through activation of signaling pathways involving Erk, Akt, and Rac.

Topics: Animals; Carotid Arteries; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Femoral Artery; Humans; Hyperplasia; Iliac Artery; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Proto-Oncogene Proteins c-akt; rac GTP-Binding Proteins; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Time Factors; Transfection; Tunica Intima; Vascular System Injuries

Gα(12/13) play a role in oncogenic transformation and tumor growth. Cysteine-rich protein 61 (CYR61) is a growth-factor-inducible angiogenic factor. In view of potential overlapping functions between Gα(12/13) and CYR61, this study investigated the role of these G proteins in CYR61 induction in association with hyperplastic vascular abnormality.. Overexpression of activated Gα(12) or Gα(13) induced CYR61 expression in vascular smooth muscle cells (VSMCs). Gene knockdown and knockout experiments revealed that sphingosine-1-phosphate (S1P) treatment induced CYR61 via Gα(12/13). JunD/activator protein-1 (AP-1) was identified as a transcription factor required for CYR61 transactivation by S1P. Deficiencies in Gα(12/13) abrogated AP-1 activation and AP-1-mediated CYR61 induction. c-Jun N-terminal kinase was responsible for CYR61 induction. Moreover, deficiencies of Gα(12/13) abolished c-Jun N-terminal kinase-dependent CYR61 induction by S1P. N-acetyl-l-cysteine or NADPH oxidase inhibitor treatment reversed CYR61 induction by S1P, indicating that reactive oxygen species are responsible for this process. The levels of Gα(12/13) were increased within thickened intimas and medias in wire-injured mouse femoral arteries, which was accompanied by simultaneous CYR61 induction. Moreover, Gα(12/13) and CYR61 were costained in the arteriosclerotic lesions immediately adjacent to human tumor tissues.. Gα(12/13) regulate AP-1-dependent CYR61 induction in VSMCs and promote VSMC migration, and they are upregulated with CYR61 in arteriosclerotic lesions.

Topics: Aged; Animals; Arteriosclerosis; Cell Movement; Cysteine-Rich Protein 61; Disease Models, Animal; Enzyme Activation; Female; GTP-Binding Protein alpha Subunits, G12-G13; HEK293 Cells; Humans; Hyperplasia; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Male; Mice; Mice, Inbred ICR; Mice, Knockout; Middle Aged; Muscle, Smooth, Vascular; Mutation; NADPH Oxidases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA Interference; Signal Transduction; Sphingosine; Transcription Factor AP-1; Transfection; Tunica Intima; Up-Regulation

Sphingosine-1-phosphate and its receptors may be involved in vascular smooth muscle cell (VSMC) proliferation following vascular injury. Here, we evaluate the effect of d-erythro-N,N-dimethylsphingosine (DMS), a sphingosine kinase (SK) inhibitor, on VSMC proliferation, apoptosis and neointimal formation.. Growth responses in vitro to fetal calf serum (FCS) were measured by [(3)H]-thymidine incorporation and extracellular signal-regulated kinase-1/2 (ERK-1/2) activation in quiescent primary cultures of porcine VSMC in the presence and absence of various concentrations of the SK inhibitor DMS. In vivo treatment with DMS was delivered with a local endoluminal catheter, following balloon injury of coronary arteries. The artery intimal formation was investigated by angiography, myography and histomorphometry.. In vitro experiments indicated that DMS induced a dose-dependent reduction in [(3)H]-thymidine incorporation and ERK-1/2 activation via a protein kinase C (PKC) independent mechanism with an IC(50) value of 12 +/- 6 and 15 +/- 10 microM respectively. DMS also reduced Akt signalling. Four weeks following in vivo delivery of DMS, complete functional endothelial regeneration was observed in all treatment groups, with significant reduction of intimal formation (vehicle 23.7 +/- 4.6% vs. DMS infusion 8.92 +/- 2.9%, P < 0.05).. Taken together, these results demonstrate that local administration of the SK inhibitor, DMS, reduced neointimal formation, and this effect could involve inhibition of ERK-1/2 and Akt signalling, and modulation of smooth muscle growth.

Topics: Animals; Apoptosis; Catheterization; Cell Proliferation; Hyperplasia; Lysophospholipids; Male; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sphingosine; Sus scrofa; Tunica Intima

About a quarter of peripheral vein grafts fail due in part to intimal hyperplasia. The proliferative capacity and response to growth inhibitors of medial smooth muscle cells and adventitial fibroblasts in vitro were studied to test the hypothesis that intrinsic differences in cells of vein grafts are associated with graft failure.. Cells were grown from explants of the medial and adventitial layers of samples of vein grafts obtained at the time of implantation. Vein graft patency and function were monitored over the first 12 months using ankle pressures and Duplex ultrasound to determine vein graft status. Cells were obtained from veins from 11 patients whose grafts remained patent (non-stenotic) and from seven patients whose grafts developed stenosis. Smooth muscle cells (SMCs) derived from media and fibroblasts derived from adventitia were growth arrested in serum-free medium and then stimulated with 1 muM sphingosine-1-phosphate (S1P), 10 nM thrombin, 10 ng/ml epidermal growth factor (EGF), 10 ng/ml platelet-derived growth factor-BB (PDGF-BB), PDGF-BB plus S1P, or PDGF-BB plus thrombin for determination of incorporation of [(3)H]-thymidine into DNA. Cells receiving PDGF-BB or thrombin were also treated with or without 100 microg/ml heparin, which is a growth inhibitor. Cells receiving thrombin were also treated with or without 150 nM AG1478, an EGF receptor kinase inhibitor.. SMCs and fibroblasts from veins of patients that developed stenosis responded more to the growth factors, such as PDGF-BB alone or in combination with thrombin or S1P, than cells from veins of patients that remained patent (P = .012). In addition, while PDGF-BB-mediated proliferation of fibroblasts from grafts that remained patent was inhibited by heparin (P < .03), PDGF-BB-mediated proliferation of fibroblasts from veins that developed stenosis was not (P > .5).. Inherent differences in the proliferative response of vein graft cells to PDGF-BB and heparin may explain, in part, the variability among patients regarding long term patency of vein grafts.

Topics: Aged; Ankle; Becaplermin; Blood Pressure; Cell Proliferation; Cells, Cultured; Constriction, Pathologic; DNA Replication; Epidermal Growth Factor; Female; Fibroblasts; Graft Occlusion, Vascular; Heparin; Humans; Hyperplasia; Lower Extremity; Lysophospholipids; Male; Middle Aged; Myocytes, Smooth Muscle; Peripheral Vascular Diseases; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-sis; Quinazolines; Saphenous Vein; Sphingosine; Thrombin; Time Factors; Tyrphostins; Ultrasonography, Doppler, Duplex; Vascular Patency

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.

Topics: Administration, Inhalation; Aniline Compounds; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokines, CC; Disease Models, Animal; Enzyme Inhibitors; Goblet Cells; Humans; Hyperplasia; Interleukins; Lysophospholipids; Mice; Mice, Inbred C57BL; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Respiratory Mucosa; Sphingosine; Thiazoles

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SphK) is the enzyme directly responsible for production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. Here we synthesized a previously unidentified SphK activator, K6PC-5, and have studied its effects on intracellular Ca(2+) signaling in HaCaT cells and epidermal differentiation in murine skin. K6PC-5, a hydrophobic compound chemically named N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide, activated SphK (obtained from C57BL/6 murine blood and F9-12 cell lysates) in a dose-dependent manner. K6PC-5 induced both intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in HaCaT cells and Ca(2+) mobilization in hairless mouse epidermis. Both dimethylsphingosine (DMS) and dihydroxysphingosine (DHS), SphK inhibitors, and transfection of SphK1-siRNA blocked K6PC-5-induced increases in [Ca(2+)](i). The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry, but independent of the classical phospholipase C-mediated pathway. In addition, K6PC-5 enhanced the expression of involucrin and filaggrin, specific differentiation-associated marker proteins in HaCaT cells, whereas transfection of SphK1-siRNA blocked the increase of involucrin. Topical K6PC-5 also enhanced the expression of involucrin, loricrin, filaggrin, and keratin 5 in intact murine epidermis. Finally, topical K6PC-5 inhibited epidermal hyperplasia by exerting antiproliferative effects on keratinocytes in murine epidermis. These results suggest that K6PC-5 acts to regulate both differentiation and proliferation of keratinocytes via [Ca(2+)](i) responses through S1P production. Thus, regulation of S1P levels may represent a novel approach for treatment of skin disorders characterized by abnormal differentiation and proliferation, such as atopic dermatitis and psoriasis.

Topics: Amides; Animals; Calcium; Calcium Signaling; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Proliferation; Enzyme Activators; Epidermis; Female; Filaggrin Proteins; Humans; Hyperplasia; Intermediate Filament Proteins; Keratin-5; Keratinocytes; Lysophospholipids; Membrane Proteins; Mice; Mice, Hairless; Mice, Inbred C57BL; Neoplasms, Germ Cell and Embryonal; Phosphotransferases (Alcohol Group Acceptor); Protein Precursors; Sphingosine; Teratocarcinoma