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.

Suppression of androgen receptor signaling induces prostate cancer migration via activation of the CCL20-CCR6 axis.

The suppression of androgen receptor (AR) expression exacerbates the migration potential of prostate cancer. This study identified a previously unrecognized regulation of the AR-controlled pathway that promotes migration potential in prostate cancer cells. Prostate cancer cells that pass through a transwell membrane (mig cells) have a higher migration potential with a decreased AR expression than parental cells. In this study, we aimed to elucidate the mechanism of migration enhancement associated with the suppression of AR signaling. Expression of C-C motif ligand 20 (CCL20) is upregulated in mig cells, unlike in the parental cells. Knockdown of AR with small interfering RNA (siAR) in LNCaP and C4-2B cells increased CCL20 secretion and enhanced the migration of cancer cells. Mig cells, CCL20-treated cells, and siAR cells promoted cell migration with an enhancement of AKT phosphorylation and Snail expression, while the addition of a C-C chemokine receptor 6 (CCR6, the specific receptor of CCL20) inhibitor, anti-CCL20 antibody, and AKT inhibitor suppressed the activation of AKT and Snail. With 59 samples of prostate cancer tissue, CCL20 secretion was profuse in metastatic cases despite low AR expression levels. Snail expression was associated with the expression of CCL20 and CCR6. A xenograft study showed that the anti-CCL20 antibody significantly inhibited Snail expression, thereby suggesting a new therapeutic approach for castration-resistant prostate cancer with the inhibition of the axis between CCL20 and CCR6.

Adipose-Derived Stem Cell Sheets Promote Meniscus Regeneration Regardless of Whether the Defect Involves the Inner Half or the Whole Width of the Anterior Half of the Medial Meniscus in a Rabbit Model.

PURPOSE: To investigate the regenerative effect of adipose-derived stem cell (ADSC) sheets in two different rabbit models of meniscal defects. METHODS: Forty-two rabbits were randomly divided into two groups: the whole (Group 1) or the inner half (Group 2) of anterior half of the medial meniscus was removed from both knees. The ADSC sheets were transplanted into one knee, whereas in the other knee the meniscal defect was left untreated (self-control). The histological score and expression of genes encoding collagen type I and II (COL1/2), SRY-box transcription factor 9 (SOX9), and aggrecan (ACAN) were compared between the ADSC sheet-treated and untreated menisci at 4 and 12 weeks. The ADSC sheet-treated menisci at 12 weeks were also analyzed immunohistochemically to assess the collagen component. RESULTS: The histological score was significantly higher in the treated side than in the control side at 4 and 12 weeks in both groups (Group 1; P = .016 and .032; Group 2; P = .030 and .016, respectively). All genes evaluated showed significantly higher expression in the treated side than in the control side in both groups, except COL2 and SOX9 at 4 weeks and COL2 at 12 weeks in Group 1, and COL1 in Group 2 at 4 weeks. The ADSC sheet-treated meniscus in Group 1 contained mostly COL1, whereas the Group 2 had less COL1, but was rich in COL2. CONCLUSIONS: ADSC sheets can promote meniscal regeneration regardless of whether the defect involves the inner half or whole width of the anterior half of the medial meniscus. However, the collagen component of the ADSC sheet-treated tissue differs depending on the defect site. CLINICAL RELEVANCE: ADSCs may help meniscal regeneration due to meniscal defects after meniscectomy. This study suggests longer-term follow-up and mechanical analysis as next steps.

Class II phosphatidylinositol 3-kinase isoforms in vesicular trafficking.

Phosphatidylinositol 3-kinases (PI3Ks) are critical regulators of many cellular processes including cell survival, proliferation, migration, cytoskeletal reorganization, and intracellular vesicular trafficking. They are a family of lipid kinases that phosphorylate membrane phosphoinositide lipids at the 3' position of their inositol rings, and in mammals they are divided into three classes. The role of the class III PI3K Vps34 is well-established, but recent evidence suggests the physiological significance of class II PI3K isoforms in vesicular trafficking. This review focuses on the recently discovered functions of the distinct PI3K-C2α and PI3K-C2ß class II PI3K isoforms in clathrin-mediated endocytosis and consequent endosomal signaling, and discusses recently reported data on class II PI3K isoforms in different physiological contexts in comparison with class I and III isoforms.

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.

Analysis of various types of single-polypeptide-chain (sc) heterodimeric A2AR/D2R complexes and their allosteric receptor-receptor interactions.

Adenosine A2A receptor (A2AR) heteromerizes with dopamine D2 receptor (D2R). However, these class A G protein-coupled receptor (GPCR) dimers are not fully formed, but depend on the equilibrium between monomer and dimer. In order to stimulate the heteromerization, we have previously shown a successful design for a fusion receptor, single-polypeptide-chain (sc) heterodimeric A2AR/D2R complex. Here, using whole cell binding assay, six more different scA2AR/D2R constructs were examined. Not only in scA2AR/D2R 'liberated' with longer spacers between the two receptors, which confer the same configuration as the prototype, the A2AR-odr4TM-D2LR, but differ in size (Forms 1-3), but also in scA2AR/D2LR (Form 6) fused with a transmembrane (TM) of another type II TM protein, instead of odr4TM, neither of their fixed stoichiometry (the apparent ratios of A2AR to D2R binding sites) was 1, suggesting their compact folding. This suggests that type II TM, either odr4 or another, facilitates the equilibrial process of the dimer formation between A2AR and D2LR, resulting in the higher-order oligomer formation from monomer of scA2AR/D2LR itself. Also, in the reverse type scA2AR/D2LR, i.e., the D2LR-odr4TM-A2AR, counter agonist-independent binding cooperativity (cooperative folding) was found to occur (Forms 4 and 5). In this way, the scA2AR/D2LR system has unveiled the cellular phenomenon as a snapshot of the molecular behavior in A2AR/D2LR dimer. Thus, these results indicate that the various designed types of functional A2AR/D2R exist even in living cells and that this fusion expression system would be useful to analyze as a model of the interaction between class A GPCRs.

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.

Collagen-Type Composition in the Semitendinosus, Quadriceps, and Patellar Tendons of a 22-Year-Old Patient: A Case Report.

Graft failure is a common postoperative complication after anterior cruciate ligament (ACL) reconstruction. Recently, a theory has emerged that histological and microstructural factors of autografts may be related to graft failure. We simultaneously collected the semitendinosus tendon (ST), quadriceps tendon (QT), and patellar tendon (PT) from a 22-year-old patient to provide insights into the differences in the collagen-type composition of the three tendons in skeletally mature patients. These findings may serve as a basis for selecting autografts for ACL to reduce graft failure rates. The patient was a 22-year-old female who required the removal of artificial ligament, screws, and washers and medial patellofemoral ligament (MPFL) reconstruction with an ST autograft after two surgeries for recurrent dislocation of the left patella. The ST, QT, and PT obtained during necessary intraoperative procedures were used as samples. The tissues were processed and immunostained; this was followed by confocal microscopy. Evaluation was performed by calculating the percentage of areas positive for collagen types I and III.The percentage of type I collagen in the ST, QT, and PT groups was 88%, 85%, and 88%, respectively.The collagen-type composition was examined following simultaneous collection of the ST, QT, and PT. The results revealed no significant differences in the content of physically strong type I collagen, which supports previous findings showing that the clinical outcomes after ACL reconstruction do not vary with the autograft used.

Differences in collagen types in the semitendinosus, quadriceps, and patellar tendons: A report using samples from an 11-year-old patient.

BACKGROUND: This study aimed to determine the differences in the proportions of types I and type III collagen in the semitendinosus tendon (ST), quadriceps tendon (QT), and patellar tendon (PT), which are frequently used as autografts for anterior cruciate ligament (ACL) reconstruction. METHODS: Orthopedic surgeons diagnosed habitual dislocation of the left patella and surgically treated an 11-year-old boy. Medial patellofemoral ligament reconstruction, medial patellar tibial ligament reconstruction, and arthroscopic lateral release were performed simultaneously. Tissue samples obtained during treatment that were no longer necessary were used as samples for this study. The samples were fixed, paraffin-embedded, and immunostained for type I and type III collagen. Stained samples were observed under a confocal microscope and evaluated visually and quantitatively to determine the percentages of type I and type III collagen. RESULTS: Visually, the ST had a higher percentage of type III collagen than the PT and QT. The QT and PT were similar in appearance; both consisted mostly of collagen type I. Quantitative evaluation using images showed that the PT comprised 100% type I collagen. The QT comprised 1% type III collagen. The ST comprised 34% type III collagen. CONCLUSION: In this patient, the QT and PT had higher percentages of type I collagen, which is considered physically strong. Type III collagen, which is considered physically weak, was most common in the ST. These factors may be associated with the high re-injury rates after ACL reconstruction using the ST for physically immature patients.

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.

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.

TGFß receptor endocytosis and Smad signaling require synaptojanin1, PI3K-C2α-, and INPP4B-mediated phosphoinositide conversions.

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.