ABSTRACT
Class II phosphatidylinositol 3-kinases (PI3K), PI3K-C2α and PI3K-C2ß, are involved in cellular processes including endocytosis, cilia formation and autophagy. However, the role of PI3K-C2α and PI3K-C2ß at the organismal level is not well understood. We found that double knockout (KO) mice with both smooth muscle-specific KO of PI3K-C2α and global PI3K-C2ß KO, but not single KO mice of either PI3K-C2α or PI3K-C2ß, exhibited reductions in arterial blood pressure and substantial attenuation of contractile responses of isolated aortic rings. In wild-type vascular smooth muscle cells, double knockdown of PI3K-C2α and PI3K-C2ß but not single knockdown of either PI3K markedly inhibited contraction with reduced phosphorylation of 20-kDa myosin light chain and MYPT1 and Rho activation, but without inhibition of the intracellular Ca2+ mobilization. These data indicate that PI3K-C2α and PI3K-C2ß play the redundant but essential role for vascular smooth muscle contraction and blood pressure regulation mainly through their involvement in Rho activation.
Subject(s)
Calcium/metabolism , Class II Phosphatidylinositol 3-Kinases/metabolism , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/physiology , rho GTP-Binding Proteins/metabolism , Animals , Blood Pressure/physiology , Cells, Cultured , Class II Phosphatidylinositol 3-Kinases/genetics , Disease Models, Animal , Isoenzymes , Mice , Mice, Knockout , Muscle Contraction/physiology , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/enzymology , rho GTP-Binding Proteins/geneticsABSTRACT
Phosphoinositide conversion regulates a diverse array of dynamic membrane events including endocytosis. However, it is not well understood which enzymes are involved in phosphoinositide conversions for receptor endocytosis. We found by small interfering RNA (siRNA)-mediated knockdown (KD) that class II PI3K α-isoform (PI3K-C2α), the 5'-phosphatase synaptojanin1 (Synj1), and the 4'-phosphatase INPP4B, but not PI3K-C2ß, Synj2, or INPP4A, were required for TGFß-induced endocytosis of TGFß receptor. TGFß induced rapid decreases in PI(4,5)P2 at the plasma membrane (PM) with increases in PI(4)P, followed by increases in PI(3,4)P2, in a TGFß receptor kinase ALK5-dependent manner. TGFß induced the recruitment of both synaptojanin1 and PI3K-C2α to the PM with their substantial colocalization. Knockdown of synaptojanin1 abolished TGFß-induced PI(4,5)P2 decreases and PI(4)P increases. Interestingly, PI3K-C2α KD abolished not only TGFß-induced PI(3,4)P2 increases but also TGFß-induced synaptojanin1 recruitment to the PM, PI(4,5)P2 decreases, and PI(4)P increases. Finally, the phosphoinositide conversions were necessary for TGFß-induced activation of Smad2 and Smad3. These observations demonstrate that the sequential phosphoinositide conversions mediated by Synj1, PI3K-C2α, and INPP4B are essential for TGFß receptor endocytosis and its signaling.
Subject(s)
Endocytosis , Nerve Tissue Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositols/metabolism , Phosphoric Monoester Hydrolases/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction , Smad Proteins/metabolism , Activin Receptors, Type II/metabolism , Cell Membrane/metabolism , Cell Nucleus/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Phosphorylation , Receptor, Transforming Growth Factor-beta Type I/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
Atherosclerosis is the major cause of ischemic coronary heart diseases and characterized by the infiltration of cholesterol-accumulating macrophages in the vascular wall. Although sphingolipids are implicated in atherosclerosis as both membrane components and lipid mediators, the precise role of sphingolipids in atherosclerosis remains elusive. Here, we found that genetic deficiency of sphingosine kinase-2 (SphK2) but not SphK1 aggravates the formation of atherosclerotic lesions in mice with ApoE deficiency. Bone marrow chimaera experiments show the involvement of SphK2 expressed in bone marrow-derived cells. In macrophages, deficiency of SphK2, a major SphK isoform in this cell type, results in increases in cellular sphingosine and ceramides. SphK2-deficient macrophages have increases in lipid droplet-containing autophagosomes and autolysosomes and defective lysosomal degradation of lipid droplets via autophagy with an impaired luminal acidic environment and proteolytic activity in the lysosomes. Transgenic overexpression of SphK1 in SphK2-deficient mice rescued aggravation of atherosclerosis and abnormalities of autophagosomes and lysosomes in macrophages with reductions of sphingosine, suggesting at least partial overlapping actions of two SphKs. Taken together, these results indicate that SphK2 is required for autophagosome- and lysosome-mediated catabolism of intracellular lipid droplets to impede the development of atherosclerosis; therefore, SphK2 may be a novel target for treating atherosclerosis.
Subject(s)
Apolipoproteins E/genetics , Atherosclerosis/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Animals , Atherosclerosis/metabolism , Atherosclerosis/pathology , Bone Marrow Cells/metabolism , Cholesterol/metabolism , Disease Models, Animal , Humans , Lipid Metabolism/genetics , Lipids/genetics , Macrophages/metabolism , Macrophages/pathology , Mice , Sphingolipids/genetics , Sphingolipids/metabolism , Sphingosine/metabolismABSTRACT
Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2ß, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2ß are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2ß. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2ß-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2ß are required for normal USM contraction and parturition mainly through their involvement in Rho activation.
Subject(s)
Class II Phosphatidylinositol 3-Kinases/metabolism , Muscle, Smooth, Vascular/enzymology , Parturition , Phosphatidylinositol 3-Kinases/metabolism , Uterine Contraction , Uterus/enzymology , rhoA GTP-Binding Protein/metabolism , Animals , Class II Phosphatidylinositol 3-Kinases/genetics , Female , Mice , Mice, Knockout , Muscle Contraction , Muscle, Smooth, Vascular/physiology , Myosin Light Chains , Phosphatidylinositol 3-Kinases/genetics , Phosphorylation , Uterus/physiology , rhoA GTP-Binding Protein/geneticsABSTRACT
Pinocytosis is an important fundamental cellular process that is used by the cell to transport fluid and solutes. Phosphoinositide 3-kinases (PI3Ks) regulate a diverse array of dynamic membrane events. However, it is not well-understood which PI3K isoforms are involved in specific mechanisms of pinocytosis. We performed knockdown studies of endogenous PI3K isoforms and clathrin heavy chain (CHC) mediated by small interfering RNA (siRNA). The results demonstrated that the class II PI3K PI3K-C2α and PI3K-C2ß, but not the class I or III PI3K, were required for pinocytosis, based on an evaluation of fluorescein-5-isothiocyanate (FITC)-dextran uptake in endothelial cells. Pinocytosis was partially dependent on both clathrin and dynamin, and both PI3K-C2α and PI3K-C2ß were required for clathrin-mediated-but not clathrin-non-mediated-FITC-dextran uptake at the step leading up to its delivery to early endosomes. Both PI3K-C2α and PI3K-C2ß were co-localized with clathrin-coated pits and vesicles. However, PI3K-C2ß, but not PI3K-C2α, was highly co-localized with actin filament-associated clathrin-coated structures and required for actin filament formation at the clathrin-coated structures. These results indicate that PI3K-C2α and PI3K-C2ß play differential, indispensable roles in clathrin-mediated pinocytosis.
Subject(s)
Class II Phosphatidylinositol 3-Kinases/metabolism , Clathrin/metabolism , Endothelial Cells/metabolism , Endothelial Cells/physiology , Phosphatidylinositol 3-Kinases/metabolism , Pinocytosis/physiology , Actin Cytoskeleton/metabolism , Animals , Cells, Cultured , Human Umbilical Vein Endothelial Cells , Humans , RNA, Small Interfering/metabolismABSTRACT
Phosphatidylinositol 3-phosphate (PI(3)P) is the predominant phosphoinositide species in early endosomes and autophagosomes, in which PI(3)P dictates traffic of these organelles. Phosphoinositide levels are tightly regulated by lipid-kinases and -phosphatases; however, a phosphatase that converts PI(3)P back to phosphatidylinositol in the endosomal and autophagosomal compartments is not fully understood. We investigated the subcellular distribution and functions of myotubularin-related protein-4 (MTMR4), which is distinct among other MTMRs in that it possesses a PI(3)P-binding FYVE domain, in lung alveolar epithelium-derived A549 cells. MTMR4 was localized mainly in late endosomes and autophagosomes. MTMR4 knockdown markedly suppressed the motility, fusion, and fission of PI(3)P-enriched structures, resulting in decreases in late endosomes, autophagosomes, and lysosomes, and enlargement of PI(3)P-enriched early and late endosomes. In amino acid- and serum-starved cells, MTMR4 knockdown decreased both autophagosomes and autolysosomes and markedly increased PI(3)P-containing autophagosomes and late endosomes, suggesting that the fusion with lysosomes of autophagosomes and late endosomes might be impaired. Notably, MTMR4 knockdown inhibited the nuclear translocation of starvation stress responsive transcription factor-EB (TFEB) with reduced expression of lysosome-related genes in starved cells. These findings indicate that MTMR4 is essential for the integrity of endocytic and autophagic pathways.
ABSTRACT
Idiopathic pulmonary fibrosis is a devastating disease with poor prognosis. The pathogenic role of the lysophospholipid mediator sphingosine-1-phosphate and its receptor S1PR2 in lung fibrosis is unknown. We show here that genetic deletion of S1pr2 strikingly attenuated lung fibrosis induced by repeated injections of bleomycin in mice. We observed by using S1pr2LacZ/+ mice that S1PR2 was expressed in alveolar macrophages, vascular endothelial cells and alveolar epithelial cells in the lung and that S1PR2-expressing cells accumulated in the fibrotic legions. Bone marrow chimera experiments suggested that S1PR2 in bone marrow-derived cells contributes to the development of lung fibrosis. Depletion of macrophages greatly attenuated lung fibrosis. Bleomycin administration stimulated the mRNA expression of the profibrotic cytokines IL-13 and IL-4 and the M2 markers including arginase 1, Fizz1/Retnla, Ccl17 and Ccl24 in cells collected from broncho-alveolar lavage fluids (BALF), and S1pr2 deletion markedly diminished the stimulated expression of these genes. BALF cells from bleomycin-administered wild-type mice showed a marked increase in phosphorylation of STAT6, a transcription factor which is activated downstream of IL-13, compared with saline-administered wild-type mice. Interestingly, in bleomycin-administered S1pr2-/- mice, STAT6 phosphorylation in BALF cells was substantially diminished compared with wild-type mice. Finally, pharmacological S1PR2 blockade in S1pr2+/+ mice alleviated bleomycin-induced lung fibrosis. Thus, S1PR2 facilitates lung fibrosis through the mechanisms involving augmentation of IL-13 expression and its signaling in BALF cells, and represents a novel target for treating lung fibrosis.
Subject(s)
Idiopathic Pulmonary Fibrosis/etiology , Interleukin-13/metabolism , Macrophages/metabolism , Receptors, Lysosphingolipid/metabolism , Animals , Bleomycin/toxicity , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Disease Models, Animal , Idiopathic Pulmonary Fibrosis/metabolism , Idiopathic Pulmonary Fibrosis/pathology , Interleukin-13/genetics , Macrophages/drug effects , Macrophages/pathology , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Lysosphingolipid/deficiency , Receptors, Lysosphingolipid/genetics , STAT6 Transcription Factor/metabolism , Signal Transduction , Sphingosine-1-Phosphate Receptors , Transplantation Chimera/genetics , Transplantation Chimera/metabolism , Up-RegulationABSTRACT
BACKGROUND: Cardiac fibroblasts, together with cardiomyocytes, occupy the majority of cells in the myocardium and are involved in myocardial remodeling. The lysophospholipid mediator sphigosine-1-phosphate (S1P) regulates functions of cardiovascular cells through multiple receptors including S1PR1-S1PR3. S1PR1 but not other S1P receptors was upregulated in angiotensin II-induced hypertrophic hearts. Therefore, we investigated a role of S1PR1 in fibroblasts for cardiac remodeling by employing transgenic mice that overexpressed S1PR1 under the control of α-smooth muscle actin promoter. In S1PR1-transgenic mouse heart, fibroblasts and/or myofibroblasts were hyperplastic, and those cells as well as vascular smooth muscle cells overexpressed S1PR1. Transgenic mice developed bi-ventricular hypertrophy by 12-week-old and diffuse interstitial fibrosis by 24-week-old without hemodynamic stress. Cardiac remodeling in transgenic mice was associated with greater ERK phosphorylation, upregulation of fetal genes, and systolic dysfunction. Transgenic mouse heart showed increased mRNA expression of angiotensin-converting enzyme and interleukin-6 (IL-6). Isolated fibroblasts from transgenic mice exhibited enhanced generation of angiotensin II, which in turn stimulated IL-6 release. Either an AT1 blocker or angiotensin-converting enzyme inhibitor prevented development of cardiac hypertrophy and fibrosis, systolic dysfunction and increased IL-6 expression in transgenic mice. Finally, administration of anti-IL-6 antibody abolished an increase in tyrosine phosphorylation of STAT3, a major signaling molecule downstream of IL-6, in the transgenic mouse heart and prevented development of cardiac hypertrophy in transgenic mice. These results demonstrate a promoting role of S1PR1 in cardiac fibroblasts for cardiac remodeling, in which angiotensin II-AT1 and IL-6 are involved.
Subject(s)
Angiotensin II/metabolism , Interleukin-6/metabolism , Receptors, Lysosphingolipid/metabolism , Signal Transduction , Angiotensin II/analysis , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Benzimidazoles/pharmacology , Benzimidazoles/therapeutic use , Biphenyl Compounds , Cardiomegaly/diagnostic imaging , Cardiomegaly/pathology , Cardiomegaly/prevention & control , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Heart Ventricles/diagnostic imaging , Humans , Interleukin-6/analysis , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myocardium/metabolism , Myocardium/pathology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Phosphorylation/drug effects , Plasmids/genetics , Plasmids/metabolism , Receptors, Lysosphingolipid/genetics , Signal Transduction/drug effects , Tetrazoles/pharmacology , Tetrazoles/therapeutic useABSTRACT
We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P1 and thereby their signaling on endosomes. TGFß, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFß1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFß-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFß receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFß receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFß receptor internalization and Smad2/3 phosphorylation. TGFß1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFß1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFß receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFß.
Subject(s)
Endocytosis/genetics , Intracellular Signaling Peptides and Proteins/genetics , Phosphatidylinositol 3-Kinases/genetics , Serine Endopeptidases/genetics , Transforming Growth Factor beta1/genetics , Animals , Endothelial Cells/metabolism , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells , Humans , Intracellular Signaling Peptides and Proteins/biosynthesis , Mice , Mice, Knockout , Serine Endopeptidases/biosynthesis , Signal Transduction , Transforming Growth Factor beta1/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
BACKGROUND: Sphingosine-1-phosphate receptor 2 (S1P(2)) is expressed in vascular endothelial cells (ECs). However, the role of S1P(2) in vascular barrier integrity and anaphylaxis is not well understood. Endothelial nitric oxide synthase (eNOS) generates nitric oxide to mediate vascular leakage, compromising survival in patients with anaphylaxis. We recently observed that endothelial S1P(2) inhibits Akt, an activating kinase of eNOS. OBJECTIVE: We tested the hypothesis that endothelial S1P(2) might suppress eNOS, exerting a protective effect against endothelial barrier disruption and anaphylaxis. METHODS: Mice deficient in S1P(2) and eNOS underwent antigen challenge or platelet-activating factor (PAF) injection. Analyses were performed to examine vascular permeability and the underlying mechanisms. RESULTS: S1pr2 deletion augmented vascular leakage and lethality after either antigen challenge or PAF injection. PAF injection induced activation of Akt and eNOS in the aortas and lungs of S1pr2-null mice, which were augmented compared with values seen in wild-type mice. Consistently, PAF-induced increase in cyclic guanosine monophosphate levels in the aorta was enhanced in S1pr-null mice. Genetic Nos3 deletion or pharmacologic eNOS blockade protected S1pr2-null mice from aggravation of barrier disruption after antigen challenge and PAF injection. ECs isolated from S1pr2-null mice exhibited greater stimulation of Akt and eNOS, with enhanced nitric oxide production in response to sphingosine-1-phosphate or PAF, compared with that seen in wild-type ECs. Moreover, S1pr2-deficient ECs showed more severe disassembly of adherens junctions with augmented S-nitrosylation of ß-catenin in response to PAF, which was restored by pharmacologic eNOS blockade. CONCLUSION: S1P(2) diminishes harmful robust eNOS stimulation and thereby attenuates vascular barrier disruption, suggesting potential usefulness of S1P(2) agonists as novel therapeutic agents for anaphylaxis.
Subject(s)
Anaphylaxis/metabolism , Nitric Oxide Synthase Type III/antagonists & inhibitors , Receptors, Lysosphingolipid/metabolism , Adherens Junctions/metabolism , Anaphylaxis/genetics , Anaphylaxis/mortality , Animals , Aorta/immunology , Aorta/metabolism , Capillary Permeability/genetics , Capillary Permeability/immunology , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Enzyme Activation , Gene Deletion , Lung/immunology , Lung/metabolism , Mice , Mice, Knockout , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/metabolism , Platelet Activating Factor/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Lysosphingolipid/genetics , Signal Transduction , beta Catenin/metabolismABSTRACT
Fibrosis is a pathological process characterized by massive deposition of extracellular matrix (ECM) such as type I/III collagens and fibronectin that are secreted by an expanded pool of myofibroblasts, which are phenotypically altered fibroblasts with more contractile, proliferative, migratory and secretory activities. Fibrosis occurs in various organs including the lung, heart, liver and kidney, resulting in loss of normal tissue architecture and functions. Myofibroblasts could originate from multiple sources including tissue-resident fibroblasts, epithelial and endothelial cells through mechanisms of epithelial/endothelial-mesenchymal transition (EMT/EndMT), and bone marrow-derived circulating progenitors called fibrocytes. Emerging evidence in recent years shows that sphingosine-1-phosphate (S1P) acts on several types of target cells and is engaged in pro-fibrotic inflammatory process and fibrogenic process through multiple mechanisms, which include vascular permeability change, leukocyte infiltration, and migration, proliferation and myofibroblast differentiation of fibroblasts. Many of these S1P actions are receptor subtype-specific. In these actions, S1P has multiple cross-talks with other cytokines, particularly transforming growth factor-ß (TGFß), which plays a major role in fibrosis. The cross-talks include the regulation of S1P production through altered expression and activity of sphingosine kinases in fibrotic lesions, altered expression of S1P receptors, and S1P receptor-mediated transactivation of TGFß signaling pathway. These cross-talks may give rise to a feed-forward, amplifying loop between S1P and TGFß, and possibly with other cytokines in stimulating fibrogenesis. Another lysophospholipid mediator lysophosphatidic acid has also been recently implicated in fibrosis. The lysophospholipid signaling pathways represent novel, promising therapeutic targets for treating refractory fibrotic diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
Subject(s)
Disease Progression , Fibrosis/metabolism , Fibrosis/pathology , Lysophospholipids/metabolism , Sphingosine/analogs & derivatives , Animals , Humans , Models, Biological , Organ Specificity , Sphingosine/metabolismABSTRACT
The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3'-phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110α and p110ß, functional roles of class II PI3Ks, comprising PI3K-C2α, PI3K-C2ß, and PI3K-C2γ, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P(1-3). We studied the roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P(1) in ECs. Knockdown of either PI3K-C2α or class I p110ß markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110α or Vps34 did not. Only p110ß was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110ß were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110ß markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110ß suppressed S1P-induced S1P(1) internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P(1) internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P(1) internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs.
Subject(s)
Class II Phosphatidylinositol 3-Kinases/physiology , Gene Expression Regulation, Enzymologic , Receptors, Lysosphingolipid/genetics , Cell Movement , Cells, Cultured , Class II Phosphatidylinositol 3-Kinases/genetics , Endocytosis , Endosomes/metabolism , Endothelial Cells/cytology , Fluorescence Resonance Energy Transfer , Human Umbilical Vein Endothelial Cells , Humans , Lysophospholipids/metabolism , RNA, Small Interfering/metabolism , Signal Transduction , Transfection , rac GTP-Binding Proteins/metabolismABSTRACT
The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell-specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate-enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly. PI3K-C2α knockdown also impaired endothelial cell signaling, including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. Together, the effects of PI3K-C2α knockdown led to defective endothelial cell migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deficiency in vivo suppressed postischemic and tumor angiogenesis and diminished vascular barrier function with a greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α has a crucial role in vascular formation and barrier integrity and represents a new therapeutic target for vascular disease.
Subject(s)
Blood-Air Barrier/metabolism , Neovascularization, Physiologic , Phosphatidylinositol 3-Kinases/metabolism , Angiotensin II/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cadherins/genetics , Cadherins/metabolism , Cell Adhesion , Cell Movement/genetics , Cell Proliferation , Cells, Cultured , Clathrin-Coated Vesicles/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mice, Knockout , Phosphatidylinositol 3-Kinases/deficiency , Phosphatidylinositol 3-Kinases/genetics , RNA Interference , RNA, Small Interfering , Receptors, Vascular Endothelial Growth Factor/metabolism , Signal Transduction/genetics , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism , trans-Golgi Network/metabolismABSTRACT
Sphingosine-1-phosphate (S1P), which acts as both the extracellular and intracellular messenger, exerts pleiotropic biological activities including regulation of formation of the vasculature, vascular barrier integrity, and lymphocyte trafficking. Many of these S1P actions are mediated by five members of the G protein-coupled S1P receptors (S1P(1) -S1P(5) ) with overlapping but distinct coupling to heterotrimeric G proteins. The biological activities of S1P are based largely on the cellular actions of S1P on migration, adhesion, and proliferation. Notably, S1P often exhibits receptor subtype-specific, bimodal effects in these cellular actions. For example, S1P(1) mediates cell migration toward S1P, that is, chemotaxis, via G(i) /Rac pathway whereas S1P(2) mediates inhibition of migration toward a chemoattractant, that is, chemorepulsion, via G(12/13) /Rho pathway, which induces Rac inhibition. In addition, S1P(1) mediates stimulation of cell proliferation through the G(i) -mediated signaling pathways including phosphatidylinositol 3-kinase (PI3K)/Akt and ERK whereas S1P(2) mediates inhibition of cell proliferation through mechanisms involving G(12/13) /Rho/Rho kinase/PTEN-dependent Akt inhibition. These differential effects of S1P receptor subtypes on migration and proliferation lead to bimodal regulation of various biological responses. An observed biological response is likely determined by an integrated outcome of the counteracting signals input by S1P receptor subtypes. More recent studies identified the new intracellular targets of S1P including the inflammatory signaling molecule TRAF2 and histone deacetylases HDAC1 and HDAC2. These interactions of S1P regulate NF-κB activity and gene expression, respectively. Development of S1P receptor agonists and antagonists with improved receptor subtype-selectivity, inhibitors, or modulators of sphingolipid-metabolizing enzymes, and their optimal drug delivery system provide novel therapeutic tactics.
Subject(s)
Lysophospholipids/metabolism , Receptors, Lysosphingolipid/metabolism , Signal Transduction , Sphingosine/analogs & derivatives , Biological Products/pharmacology , Biological Products/therapeutic use , Cell Proliferation , Chemotaxis , Gene Expression Regulation/drug effects , Humans , NF-kappa B/genetics , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Lysosphingolipid/agonists , Receptors, Lysosphingolipid/antagonists & inhibitors , Receptors, Lysosphingolipid/genetics , Signal Transduction/drug effects , Sphingosine/metabolism , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/metabolism , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolismABSTRACT
Sphingosine-1-phosphate (S1P) is a plasma lipid mediator with multiple roles in mammalian development, physiology and pathophysiology. It is constitutively produced mostly by erythrocytes by the action of sphingosine kinase 1 (SphK1), resulting in high (â¼0.5 micromolar) steady-state plasma S1P content and steep S1P concentration gradient imposed between plasma/lymph/tissue interstitial fluid. S1P is also locally produced by activated platelets and tumor cells, in the latter case SphK1 is a downstream target of activated Ras mutant and hypoxia, and is frequently upregulated especially in advanced stages of tumors. Most if not all of the S1P actions in vertebrates are mediated through evolutionarily conserved G protein-coupled S1P receptor family. Ubiquitously expressed mammalian subtypes S1PR1, S1PR2 and S1PR3 mediate pleiotropic actions of S1P in diverse cell types, through coupling to distinctive repertoire of heterotrimeric G proteins. S1PR1 and S1PR3 mediate directed cell migration toward S1P through coupling to G(i) and activating Rac, a Rho family small G protein essential for cell migration. Indeed, S1PR1 expressed in lymphocytes directs their egress from lymph nodes into lymph and recirculation, serving as the target for downregulation by the immunosuppressant FTY720 (fingolimod). S1PR1 in endothelial cells plays an essential role in vascular maturation in embryonic stage, and mediates angiogenic and vascular protective roles of S1P which include eNOS activation and maintenance of barrier integrity. It is likely that S1PR1 and SphK1 expressed in host endothelial cells and tumor cells act in concert in a paracrine loop to contribute to tumor angiogenesis, tumor invasion and progression. In sharp contrast, S1PR2 mediates S1P inhibition of Rac at the site downstream of G(12/13)-mediated Rho activation, thus identified as the first G protein-coupled receptor that negatively regulates Rac and cell migration. S1PR2 could also mediate inhibition of Akt and cell proliferation/survival signaling via Rho-ROCK-PTEN pathway. S1PR2 expressed in tumor cells mediates inhibition of cell migration and invasion in vitro and metastasis in vivo. Moreover, S1PR2 expressed in host endothelial cells and tumor-infiltrating myeloid cells in concert mediates potent inhibition of tumor angiogenesis and tumor growth in vivo, with inhibition of VEGF expression and MMP9 activity. These recent findings provide further basis for S1P receptor subtype-specific, novel therapeutic tactics for individualized treatment of patients with cancer.
Subject(s)
Cardiovascular System/metabolism , Lysophospholipids/metabolism , Signal Transduction , Sphingosine/analogs & derivatives , Animals , Humans , Lysophospholipids/chemistry , Neovascularization, Physiologic , Receptors, Lysosphingolipid/metabolism , Sphingosine/chemistry , Sphingosine/metabolismABSTRACT
Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that has pleiotropic effects in a variety of cell types including ECs, SMCs, and macrophages, all of which are central to the development of atherosclerosis. It may therefore exert stimulatory and inhibitory effects on atherosclerosis. Here, we investigated the role of the S1P receptor S1PR2 in atherosclerosis by analyzing S1pr2-/- mice with an Apoe-/- background. S1PR2 was expressed in macrophages, ECs, and SMCs in atherosclerotic aortas. In S1pr2-/-Apoe-/- mice fed a high-cholesterol diet for 4 months, the area of the atherosclerotic plaque was markedly decreased, with reduced macrophage density, increased SMC density, increased eNOS phosphorylation, and downregulation of proinflammatory cytokines compared with S1pr2+/+Apoe-/- mice. Bone marrow chimera experiments indicated a major role for macrophage S1PR2 in atherogenesis. S1pr2-/-Apoe-/- macrophages showed diminished Rho/Rho kinase/NF-κB (ROCK/NF-κB) activity. Consequently, they also displayed reduced cytokine expression, reduced oxidized LDL uptake, and stimulated cholesterol efflux associated with decreased scavenger receptor expression and increased cholesterol efflux transporter expression. S1pr2-/-Apoe-/- ECs also showed reduced ROCK and NF-κB activities, with decreased MCP-1 expression and elevated eNOS phosphorylation. Pharmacologic S1PR2 blockade in S1pr2+/+Apoe-/- mice diminished the atherosclerotic plaque area in aortas and modified LDL accumulation in macrophages. We conclude therefore that S1PR2 plays a critical role in atherogenesis and may serve as a novel therapeutic target for atherosclerosis.
Subject(s)
Apolipoproteins E/deficiency , Atherosclerosis/immunology , Atherosclerosis/pathology , Macrophages/immunology , Receptors, Lysosphingolipid/metabolism , Animals , Apolipoproteins E/genetics , Atherosclerosis/genetics , Becaplermin , Cells, Cultured , Female , Lysophospholipids/metabolism , Macrophages/cytology , Macrophages/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/physiology , NF-kappa B/genetics , NF-kappa B/metabolism , Plaque, Atherosclerotic/pathology , Platelet-Derived Growth Factor/metabolism , Proto-Oncogene Proteins c-sis , Receptors, Lysosphingolipid/antagonists & inhibitors , Receptors, Lysosphingolipid/genetics , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Sphingosine-1-Phosphate Receptors , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolismABSTRACT
Therapeutic angiogenesis is a promising strategy for treating ischemia. The lysophospholipid mediator sphingosine-1-phosphate (S1P) acts on vascular endothelial cells to stimulate migration and tube formation, and plays the critical role in developmental angiogenesis. We developed poly(lactic-co-glycolic-acid) (PLGA)-based S1P-containing microparticles (PLGA-S1P), which are biodegradable and continuously release S1P, and studied the effects of PLGA-S1P on neovascularization in murine ischemic hindlimbs. Intramuscular injections of PLGA-S1P stimulated blood flow in C57BL/6 mice dose-dependently, with repeated administrations at a 3-day interval, rather than a single bolus or 6-day interval, over 28 days conferring the optimal stimulating effect. In Balb/c mice that exhibit limb necrosis and dysfunction due to retarded blood flow recovery, injections of PLGA-S1P stimulated blood flow with alleviation of limb necrosis and dysfunction. PLGA-S1P alone did not induce edema in ischemic limbs, and rather blocked vascular endothelial growth factor-induced edema. PLGA-S1P not only increased the microvessel densities in ischemic muscle, but promoted coverage of vessels with smooth muscle cells and pericytes, thus stabilizing vessels. PLGA-S1P stimulated Akt and ERK with increased phosphorylation of endothelial nitric oxide synthase in ischemic muscle. The effects of the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methylester, showed that PLGA-S1P-induced blood flow stimulation was partially dependent on nitric oxide. Injections of PLGA-S1P also increased the expression of angiogenic factors and the recruitment of CD45-, CD11b- and Gr-1-positive myeloid cells, which are implicated in post-ischemic angiogenesis, into ischemic muscle. These results indicate that PLGA-based, sustained local delivery of S1P is a potentially useful therapeutic modality for stimulating post-ischemic angiogenesis.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/physiology , Ischemia/drug therapy , Ischemia/physiopathology , Lactic Acid/administration & dosage , Lysophospholipids/administration & dosage , Neovascularization, Physiologic/drug effects , Nitric Oxide Synthase Type III/physiology , Polyglycolic Acid/administration & dosage , Proto-Oncogene Proteins c-akt/physiology , Sphingosine/analogs & derivatives , Animals , Delayed-Action Preparations/administration & dosage , Disease Models, Animal , Hindlimb/blood supply , Hindlimb/drug effects , Ischemia/enzymology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Microspheres , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/enzymology , Neovascularization, Pathologic/physiopathology , Neovascularization, Physiologic/physiology , Polylactic Acid-Polyglycolic Acid Copolymer , Random Allocation , Regional Blood Flow/drug effects , Regional Blood Flow/physiology , Sphingosine/administration & dosageABSTRACT
Sphingosine-1-phosphate (S1P) has been implicated in tumor angiogenesis by acting through the G(i)-coupled chemotactic receptor S1P(1). Here, we report that the distinct receptor S1P(2) is responsible for mediating the G(12/13)/Rho-dependent inhibitory effects of S1P on Akt, Rac, and cell migration, thereby negatively regulating tumor angiogenesis and tumor growth. By using S1P(2)(LacZ/+) mice, we found that S1P(2) was expressed in both tumor and normal blood vessels in many organs, in both endothelial cells (EC) and vascular smooth muscle cells, as well as in tumor-associated, CD11b-positive bone marrow-derived cells (BMDC). Lewis lung carcinoma or B16 melanoma cells implanted in S1P(2)-deficient (S1P(2)(-/-)) mice displayed accelerated tumor growth and angiogenesis with enhanced association of vascular smooth muscle cells and pericytes. S1P(2)(-/-) ECs exhibited enhanced Rac activity, Akt phosphorylation, cell migration, proliferation, and tube formation in vitro. Coinjection of S1P(2)(-/-) ECs and tumor cells into wild-type mice also produced a relative enhancement of tumor growth and angiogenesis in vivo. S1P(2)(-/-) mice were also more efficient at recruiting CD11b-positive BMDCs into tumors compared with wild-type siblings. Bone marrow chimera experiments revealed that S1P(2) acted in BMDCs to promote tumor growth and angiogenesis. Our results indicate that, in contrast to endothelial S1P(1), which stimulates tumor angiogenesis, S1P(2) on ECs and BMDCs mediates a potent inhibition of tumor angiogenesis, suggesting a novel therapeutic tactic for anticancer treatment.