Discoidin domain receptor 2 signaling through PIK3C2α in fibroblasts promotes lung fibrosis.

Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), portends significant morbidity and mortality, and current therapeutic options are suboptimal. We have previously shown that type I collagen signaling through discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase expressed by fibroblasts, is critical for the regulation of fibroblast apoptosis and progressive fibrosis. However, the downstream signaling pathways for DDR2 remain poorly defined and could also be attractive potential targets for therapy. A recent phosphoproteomic approach indicated that PIK3C2α, a poorly studied member of the PI3 kinase family, could be a downstream mediator of DDR2 signaling. We hypothesized that collagen I/DDR2 signaling through PIK3C2α regulates fibroblast activity during progressive fibrosis. To test this hypothesis, we found that primary murine fibroblasts and IPF-derived fibroblasts stimulated with endogenous or exogenous type I collagen led to the formation of a DDR2/PIK3C2α complex, resulting in phosphorylation of PIK3C2α. Fibroblasts treated with an inhibitor of PIK3C2α or with deletion of PIK3C2α had fewer markers of activation after stimulation with TGFß and more apoptosis after stimulation with a Fas-activating antibody. Finally, mice with fibroblast-specific deletion of PIK3C2α had less fibrosis after bleomycin treatment than did littermate control mice with intact expression of PIK3Cα. Collectively, these data support the notion that collagen/DDR2/PIK3C2α signaling is critical for fibroblast function during progressive fibrosis, making this pathway a potential target for antifibrotic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Sphingosine kinase 1 is involved in triglyceride breakdown by maintaining lysosomal integrity in brown adipocytes.

Sphingosine 1-phosphate (S1P) has been implicated in brown adipose tissue (BAT) formation and energy consumption; however, the mechanistic role of sphingolipids, including S1P, in BAT remains unclear. Here, we showed that, in mice, BAT activation by cold exposure upregulated mRNA and protein expression of the S1P-synthesizing enzyme sphingosine kinase 1 (SphK1) and S1P production in BAT. Treatment of wild-type brown adipocytes with exogenous S1P or S1P receptor subtype-selective agonists stimulated triglyceride (TG) breakdown only marginally, compared with noradrenaline. However, genetic deletion of Sphk1 resulted in hypothermia and diminished body weight loss upon cold exposure, suggesting that SphK1 is involved in thermogenesis through mechanisms different from receptor-mediated, extracellular action of S1P. In BAT of wild-type mice, SphK1 was localized largely in the lysosomes of brown adipocytes. In the brown adipocytes of Sphk1-/- mice, the number of lysosomes was reduced and lysosomal function, including proteolytic activity, acid esterase activity, and motility, was impaired. Concordantly, nuclear translocation of transcription factor EB, a master transcriptional regulator of lysosome biogenesis, was reduced, leading to decreased mRNA expression of the lysosome-related genes in Sphk1-/- BAT. Moreover, BAT of Sphk1-/- mice showed greater TG accumulation with dominant larger lipid droplets in brown adipocytes. Inhibition of lysosomes with chloroquine resulted in a less extent of triglyceride accumulation in Sphk1-/- brown adipocytes compared with wild-type brown adipocytes, suggesting a reduced lysosome-mediated TG breakdown in Sphk1-/- mice. Our results indicate a novel role of SphK1 in lysosomal integrity, which is required for TG breakdown and thermogenesis in BAT.

Pancreas lineage allocation and specification are regulated by sphingosine-1-phosphate signalling.

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches.

Everolimus-Eluting Biodegradable Abluminal Coating Stent versus Durable Conformal Coating Stent: Termination of the Inflammatory Response Associated with Neointimal Healing in a Porcine Coronary Model.

OBJECTIVES: We evaluated the effect of the different carrier systems on early vascular response through histological analysis and scanning electron microscopy using a porcine model. BACKGROUND: Although Synergy™ and Promus PREMIER™ share an identical stent material and drug elution (everolimus), they use different drug carrier systems: biodegradable abluminal coating polymer or durable conformal coating polymer, respectively. However, data regarding the impact of the different coating systems on vessel healing are currently limited. METHODS: Twelve Synergy™ and Promus PREMIER™ were implanted in 12 swine. Histopathological analysis of the stented segments was performed on the 2nd and 14th days after implantation. Morphometric analysis of the inflammation and intimal fibrin content was also performed. RESULTS: On the 2nd day, neointimal thickness, percentage of neointimal area, and inflammatory and intimal fibrin content scores were not significantly different between the two groups. On the 14th day, the inflammatory and intimal fibrin content scores were significantly lower in Synergy™ versus those observed in Promus PREMIER™. In Synergy™, smooth muscle cells were found and the neointimal layers were smooth. In contrast, inflammatory cells were observed surrounding the struts of Promus PREMIER™. CONCLUSIONS: These results demonstrate that termination of reactive inflammation is accelerated after abluminal coating stent versus implantation of conformal coating stent.

Sphigosine-1-phosphate receptor 1 promotes neointimal hyperplasia in a mouse model of carotid artery injury.

Vascular remodeling, resulting from proliferation and migration of vascular smooth muscle cells (VSMCs), is a major cause of atherosclerosis and restenosis. The lysophospholipid mediator sphingosine-1-phosphate (S1P) regulates proliferation and migration of VSMCs via S1P-specific G protein-coupled receptors, including S1P receptor 1 (S1PR1) to S1PR3. However, the role of S1PR1 in vascular remodeling is not well understood. Therefore, in this study, we aimed to investigate the effect of S1PR1 on neointimal hyperplasia in a carotid artery ligation mouse model using transgenic C57Bl/6 mice that overexpressed S1PR1 (Tg-S1PR1) under the control of α-smooth muscle actin promoter. We found that S1PR1 expression in carotid artery was upregulated after carotid artery ligation in non-transgenic (nTg) mice. Tg-S1PR1 mice showed enhanced ligation-induced neointimal hyperplasia with increased neointimal cell proliferation, compared with control nTg mice. VSMCs isolated from Tg-S1PR1 mice showed enhanced proliferation and migration in response to S1P stimulation. VSMCs from Tg-S1PR1 mice showed greater expression of interleukin-6 (IL-6) compared with nTg mouse-derived VSMCs, and administration of IL-6-neutralizing antibody into Tg-S1PR1 mice suppressed neointimal hyperplasia. These results suggest that S1P-S1PR1 signaling plays an important role in neointimal hyperplasia after vascular injury via IL-6 production.

MTMR4, a phosphoinositide-specific 3'-phosphatase, regulates TFEB activity and the endocytic and autophagic pathways.

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.

Early endothelialization associated with a biolimus A9 bioresorbable polymer stent in a porcine coronary model.

Although Nobori®, with a bioresorbable polymer and biolimus A9 abluminal coating, has unique characteristics, few data exist regarding endothelialization early after implantation. Fifteen Nobori® and 14 control bare-metal stents (S-stent™) were implanted in 12 pigs. Histopathology of stented segments, inflammation, and intimal fibrin content was evaluated on the 2nd and 14th day after implantation. On the 2nd day, endothelial cells were morphologically and immunohistologically confirmed on the surface of both stents, although some inflammatory cells might be involved. Stent surface endothelialization evaluated with a scanning electron microscope showed partial cellular coverage in both stents. On the 14th day, neointimal thickness and percentage of the neointimal area were significantly lower in Nobori® than in S-stent™ (51.4 ± 4.5 vs. 76.4 ± 23.6 µm, p < 0.05 and 10.8 ± 2.6 vs. 14.1 ± 4.2%, p < 0.01). No significant differences were found in these parameters on the 2nd day (17.3 ± 14.9 vs. 26.7 ± 13.6 µm and 3.7 ± 3.0 vs. 6.7 ± 3.7%), in inflammatory and intimal fibrin content scores. These results demonstrate that endothelialization could occur early after Nobori® implantation with similar inflammatory reaction to bare-metal stents, probably contributing to low frequency of in-stent thrombosis and restenosis.

Phosphatidylinositol 3-kinase class II α-isoform PI3K-C2α is required for transforming growth factor ß-induced Smad signaling in endothelial cells.

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.

[Inhibitory Effects of Sphingosine-1-phosphate Receptor-2 on Vascular Permeability in Mice].

OBJECTIVES: To determine the effect of sphingosine-1-phosphate receptor 2 (S1PR2) on vascular permeability in mice. METHODS: Acute lung injury models of mice were constructed with intra-tracheal administration of lipopolysaccharide (LPS) and compared with the controls with intra-tracheal administration of saline. The effect of S1PR2 on vascular permeability was observed by detecting leakage of Evans blue into lung tissues, pulmonary vascular leakage of fluorescein isothiocyanate (FITC)-dextran, and the wet/dry mass ratio of lungs. The effect of vascular endothelial growth factor (VEGF) on vascular endothelial permeability was detected by Miles analysis. RESULTS: LPS injections induced significant Evans blue leakage, FITC-dextran pulmonary vascular leakage and pulmonary edema, which appeared to be more serious in S1PR2-deleted mice compared with those in wild-type mice. LPS enhanced Evans blue leakage associated with VEGF in a dose-dependent way in both S1PR2-deleted mice and wild type mice. But the vascular permeability response in subcutaneous tissues induced by VEGF was higher in S1PR2-deleted mice than that in wild-type mice. CONCLUSIONS: S1PR2 is involved in endothelial cell barrier protections, which inhibits vascular permeability.

Selenoprotein P as a diabetes-associated hepatokine that impairs angiogenesis by inducing VEGF resistance in vascular endothelial cells.

AIMS/HYPOTHESIS: Impaired angiogenesis induced by vascular endothelial growth factor (VEGF) resistance is a hallmark of vascular complications in type 2 diabetes; however, its molecular mechanism is not fully understood. We have previously identified selenoprotein P (SeP, encoded by the SEPP1 gene in humans) as a liver-derived secretory protein that induces insulin resistance. Levels of serum SeP and hepatic expression of SEPP1 are elevated in type 2 diabetes. Here, we investigated the effects of SeP on VEGF signalling and angiogenesis. METHODS: We assessed the action of glucose on Sepp1 expression in cultured hepatocytes. We examined the actions of SeP on VEGF signalling and VEGF-induced angiogenesis in HUVECs. We assessed wound healing in mice with hepatic SeP overexpression or SeP deletion. The blood flow recovery after ischaemia was also examined by using hindlimb ischaemia model with Sepp1-heterozygous-knockout mice. RESULTS: Treatment with glucose increased gene expression and transcriptional activity for Sepp1 in H4IIEC hepatocytes. Physiological concentrations of SeP inhibited VEGF-stimulated cell proliferation, tubule formation and migration in HUVECs. SeP suppressed VEGF-induced reactive oxygen species (ROS) generation and phosphorylation of VEGF receptor 2 (VEGFR2) and extracellular signal-regulated kinase 1/2 (ERK1/2) in HUVECs. Wound closure was impaired in the mice overexpressing Sepp1, whereas it was improved in SeP (-/-)mice. SeP (+/-)mice showed an increase in blood flow recovery and vascular endothelial cells after hindlimb ischaemia. CONCLUSIONS/INTERPRETATION: The hepatokine SeP may be a novel therapeutic target for impaired angiogenesis in type 2 diabetes.

Essential role of class II phosphatidylinositol-3-kinase-C2α in sphingosine 1-phosphate receptor-1-mediated signaling and migration in endothelial cells.

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.

Sphingosine-1-phosphate as a mediator involved in development of fibrotic diseases.

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.

Sphingosine-1-phosphate receptor 2 protects against anaphylactic shock through suppression of endothelial nitric oxide synthase in mice.

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.

Antagonism of sphingosine 1-phosphate receptor 2 causes a selective reduction of portal vein pressure in bile duct-ligated rodents.

UNLABELLED: Sinusoidal vasoconstriction, in which hepatic stellate cells operate as contractile machinery, has been suggested to play a pivotal role in the pathophysiology of portal hypertension. We investigated whether sphingosine 1-phosphate (S1P) stimulates contractility of those cells and enhances portal vein pressure in isolated perfused rat livers with Rho activation by way of S1P receptor 2 (S1P(2) ). Rho and its effector, Rho kinase, reportedly contribute to the pathophysiology of portal hypertension. Thus, a potential effect of S1P(2) antagonism on portal hypertension was examined. Intravenous infusion of the S1P(2) antagonist, JTE-013, at 1 mg/kg body weight reduced portal vein pressure by 24% without affecting mean arterial pressure in cirrhotic rats induced by bile duct ligation at 4 weeks after the operation, whereas the same amount of S1P(2) antagonist did not alter portal vein pressure and mean arterial pressure in control sham-operated rats. Rho kinase activity in the livers was enhanced in bile duct-ligated rats compared to sham-operated rats, and this enhanced Rho kinase activity in bile duct-ligated livers was reduced after infusion of the S1P(2) antagonist. S1P(2) messenger RNA (mRNA) expression, but not S1P(1) or S1P(3) , was increased in bile duct-ligated livers of rats and mice and also in culture-activated rat hepatic stellate cells. S1P(2) expression, determined in S1P 2LacZ/+ mice, was highly increased in hepatic stellate cells of bile duct-ligated livers. Furthermore, the increase of Rho kinase activity in bile duct-ligated livers was observed as early as 7 days after the operation in wildtype mice, but was less in S1P 2-/- mice. CONCLUSION: S1P may play an important role in the pathophysiology of portal hypertension with Rho kinase activation by way of S1P(2) . The S1P(2) antagonist merits consideration as a novel therapeutic agent for portal hypertension.

CCL2/CCR2 augments the production of transforming growth factor-beta1, type 1 collagen and CCL2 by human CD45-/collagen 1-positive cells under high glucose concentrations.

BACKGROUND: The migration and activation of circulating profibrotic cells including fibrocytes by the action of the chemokine/chemokine receptor system has been implicated in pathological fibrogenesis. In the present study, the involvement of collagen 1 (Col1)-producing cells, CD45-positive/collagen-1-positive (CD45(+)/Col1(+)) cells originally named as fibrocytes via CC chemokine receptor 2 (CCR2), a cognate receptor of CCL2/monocyte chemoattractant protein, was examined in diabetic conditions. METHODS: Human CD45(+)/Col1(+) cells originating from the peripheral blood of healthy volunteers were incubated with high concentrations of D-glucose or D-mannitol as an osmotic control for 12, 24 or 48 h. In addition, these cells were preincubated with CCL2 under high glucose concentrations. We also examined the effects of the inhibitors of glucose transporters (GLUTs), reactive oxygen species or CCR2 on the expression of transforming growth factor beta1 (TGF-ß1), pro-α1 chain of Col1 (COL1A1), and CCL2. RESULTS: Stimulation of CD45(+)/Col1(+) cells with high glucose concentrations increased the mRNA and protein levels of TGF-ß1 and CCL2 and those of pro-COL1A1, and this effect was mediated in part by increased osmolality. Preincubation of the cells with cytochalasin B (a GLUT inhibitor) or N-acetylcysteine (an antioxidant) blocked the stimulatory effect of high glucose concentrations on these profibrotic molecules. In addition, preincubation of the cells with CCL2 enhanced the high glucose-induced upregulation of TGF-ß1, pro-COL1A1 and CCL2 and migration of the cells, and this effect was partly inhibited by treatment with CCR2 inhibitors. CONCLUSION: These results suggest that CD45(+)/Col1(+) cells may be directly involved, in part through CCL2/CCR2 signaling, in the fibrotic process under diabetic conditions.

Roles of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor 2 in Endotoxin-Induced Acute Retinal Inflammation.

PURPOSE: To investigate the roles of sphingosine kinases (SphKs) and sphingosine-1-phosphate receptors (S1PRs) in endotoxin-induced uveitis (EIU) mice. METHODS: EIU model was induced using an intraperitoneal injection of lipopolysaccharide (LPS). The expression of SphKs and S1PRs in the retina was assessed using quantitative polymerase chain reaction (qPCR) and immunofluorescence. The effects of S1PR antagonists on the expression of inflammatory cytokines in the retina were evaluated using qPCR and western blotting. Effects of leukocyte infiltration of the retinal vessels were evaluated to determine the effects of the S1PR2 antagonist and genetic deletion of S1PR2 on retinal inflammation. RESULTS: Retinal SphK1 expression was significantly upregulated in EIU. SphK1 was expressed in the GCL, IPL, and OPL and S1PR2 was expressed in the GCL, INL, and OPL. Positive cells in IPL and OPL of EIU retina were identified as endothelial cells. S1PR2 antagonist and genetic deletion of S1PR2 significantly suppressed the expression of IL-1α, IL-6, TNF-α, and ICAM-1, whereas S1PR1/3 antagonist did not. Use of S1PR2 antagonist and S1PR2 knockout in mice significantly ameliorated leukocyte adhesion induced by LPS. CONCLUSION: SphK1/S1P/S1PR2 signaling was upregulated in EIU and S1PR2 inhibition suppressed inflammatory response. Targeting this signaling pathway has potential for treating retinal inflammatory diseases.

The ATG5 interactome links clathrin-mediated vesicular trafficking with the autophagosome assembly machinery.

Autophagosome formation involves the sequential actions of conserved ATG proteins to coordinate the lipidation of the ubiquitin-like modifier Atg8-family proteins at the nascent phagophore membrane. Although the molecular steps driving this process are well understood, the source of membranes for the expanding phagophore and their mode of delivery are only now beginning to be revealed. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12-ATG5 conjugate, a crucial player during Atg8-family protein lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins alters autophagic flux in cell-based starvation assays. To determine whether the ATG12-ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. We report changes in the relative proportions of individual cell surface proteins and show that cell surface levels of the SLC7A5-SLC3A2 amino acid transporter are influenced by autophagy capability. Our data provide evidence for direct regulatory coupling between the ATG12-ATG5 conjugate and the clathrin membrane trafficking system and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery. Abbreviations: ATG, autophagy related; BafA1, bafilomycin A1; GFP, green fluorescent protein; HIP1R, huntingtin interacting protein 1 related; MEF, mouse embryo fibroblast; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha; SILAC, stable isotope labelling with amino acids in culture; SQSTM1, sequestosome 1; STRING, search tool for the retrieval of interacting genes/proteins.

Involvement of membrane type 1-matrix metalloproteinase (MT1-MMP) in RAGE activation signaling pathways.

An advanced glycation end products (AGE)/a receptor for AGE (RAGE) axis plays a key role in diabetic vascular complications. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been shown to function not only as a proteolytic enzyme but also as a signaling molecule. In this study, we investigated the role of MT1-MMP in the AGE/RAGE-triggered signaling pathways in cultured rabbit smooth muscle cells (SMCs) and the molecular interaction between RAGE and MT1-MMP in vitro and in vivo. In SMCs, AGE-activated Rac1 and p47(phox) within 1 min, NADPH oxidase activity and reactive oxygen species (ROS) generation within 5 min, and NF-κB phosphorylation within 15 min, thereby inducing redox-sensitive molecular expression. Silencing of RAGE by small-interfering RNA (siRNA) blocked the AGE-induced signaling pathways. AGE-induced geranylgeranyl transferase I (GGTase I) activity, Rac1·p47(phox) activation, NADPH oxidase activity, ROS generation, and molecular expression were also markedly attenuated by silencing of MT1-MMP. An inhibitor of GGTase I mimicked the effects of MT1-MMP-specific siRNA. Fluorescent immunohistochemistry revealed that MT1-MMP was partially co-localized with RAGE in SMCs, and RAGE was found to form a complex with MT1-MMP in both cultured SMCs and the aortae of diabetic rats by immunoprecipitation. Furthermore, MT1-MMP and RAGE formed a complex in the aortic atherosclerotic lesions of hyperlipidemic rabbits. We show that MT1-MMP plays a crucial role in RAGE-activated NADPH oxidase-dependent signaling pathways and forms a complex with RAGE in the vasculature, thus suggesting that MT1-MMP may be a novel therapeutic target for diabetic vascular complications.

Similar changes of hypothalamic feeding-regulating peptides mRNAs and plasma leptin levels in PTHrP-, LIF-secreting tumors-induced cachectic rats and adjuvant arthritic rats.

Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia in malignancy. However, it is difficult to explain the mechanism of anorexia/cachexia with PTHrP secretion in detail. Previously, we demonstrated that the expressions of orexigenic peptides increased and anorexigenic peptides decreased under cachectic conditions in rats carrying tumors secreting PTHrP. In this study, we investigated whether such changes in the expression of hypothalamic feeding-regulating peptides can be solely attributed to PTHrP or are a general response under cachectic conditions. Cachectic syndromes were induced in rats by: (i) inoculation of human lung cancer LC-6 cells that secreted PTHrP, (ii) inoculation of human melanoma SEKI cells that secrete not PTHrP but LIF1, (iii) injection of heat-killed Mycobacterium leading to arthritis (AA) and (iv) oral administration of a high dose of 1α,25(OH)(2)D(3) that resulted in hypercalcemia. The LC-6-bearing rats and AA rats were treated with or without anti-PTHrP antibody and indomethacin, respectively, and the expression of the hypothalamic feeding-regulating peptide mRNAs were examined by in situ hybridization histochemistry. The orexigenic peptide mRNAs, such as neuropeptide Y and agouti-related protein, were significantly increased, and that of anorexigenic peptide mRNAs, such as proopiomelanocortin, cocaine- and amphetamine-regulated transcript and corticotropin-releasing hormone were significantly decreased when they developed cachectic syndromes and AA. A high dose of 1α,25(OH)(2)D(3) caused hypercalcemia and body weight loss but did not affect the expression of hypothalamic feeding-regulating peptide mRNAs. The expressions of the hypothalamic feeding-regulating peptides change commonly in different chronic cachectic models without relating to serum calcium levels.

Class II phosphatidylinositol 3-kinase-C2α is essential for Notch signaling by regulating the endocytosis of γ-secretase in endothelial cells.

The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) plays a crucial role in angiogenesis at least in part through participating in endocytosis and, thereby, endosomal signaling of several cell surface receptors including VEGF receptor-2 and TGFß receptor in vascular endothelial cells (ECs). The Notch signaling cascade regulates many cellular processes including cell proliferation, cell fate specification and differentiation. In the present study, we explored a role of PI3K-C2α in Delta-like 4 (Dll4)-induced Notch signaling in ECs. We found that knockdown of PI3K-C2α inhibited Dll4-induced generation of the signaling molecule Notch intracellular domain 1 (NICD1) and the expression of Notch1 target genes including HEY1, HEY2 and NOTCH3 in ECs but not in vascular smooth muscle cells. PI3K-C2α knockdown did not inhibit Dll4-induced endocytosis of cell surface Notch1. In contrast, PI3K-C2α knockdown as well as clathrin heavy chain knockdown impaired endocytosis of Notch1-cleaving protease, γ-secretase complex, with the accumulation of Notch1 at the perinuclear endolysosomes. Pharmacological blockage of γ-secretase also induced the intracellular accumulation of Notch1. Taken together, we conclude that PI3K-C2α is required for the clathrin-mediated endocytosis of γ-secretase complex, which allows for the cleavage of endocytosed Notch1 by γ-secretase complex at the endolysosomes to generate NICD1 in ECs.