Involvement of sphingosine 1-phosphate signaling in insulin-like growth factor-II/mannose 6-phosphate receptor trafficking from endosome to the trans-Golgi network.

The insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6P) receptor is a multifunctional glycoprotein not only play roles in IGF-II degradation and pro-TGFß activation but binding to and transport M6P-bearing lysosomal enzymes from the trans-Golgi network (TGN) or the cell surface to lysosomes. At present, information regarding a retrograde transport of IGF-II/M6P receptor from endosomes to the TGN is still limited. We show here that a continuous ligand-dependent activation of sphingosine 1-phosphate receptor type 3 (S1P3R) on the endosomal membranes is required for subsequent recycling back of cargo-unloaded IGF-II/M6P receptors to the TGN. We have further clarified that Gq coupled with S1P3R plays a critical role in the activation of casein kinase 2, which phosphorylates and keeps PACS1 connector protein active for the association with IGF-II/M6P receptors, which enables transport carrier formation with the aid of other adaptor proteins toward the TGN. These findings shed light on the molecular mechanism underlying how continuous activation of the S1P receptor and subsequent downstream Gq signaling regulates the retrograde transport of the empty IGF-II/M6P receptors back to the TGN.

Extracellular α-synuclein impairs sphingosine 1-phosphate receptor type 3 (S1PR3)-regulated lysosomal delivery of cathepsin D in HeLa cells.

α-Synuclein (α-Syn)-positive intracellular fibrillar protein deposits, known as Lewy bodies, are thought to be involved in the pathogenesis of Parkinson's disease (PD). Although recent lines of evidence suggested that extracellular α-Syn secreted from pathogenic neurons contributes to the propagation of PD pathology, the precise mechanism of action remains unclear. We have reported that extracellular α-Syn caused sphingosine 1-phosphate (S1P) receptor type 1 (S1PR1) uncoupled from Gi and inhibited downstream G-protein signaling in SH-SY5Y cells, although its patho/physiological role remains to be clarified. Here we show that extracellular α-Syn caused S1P receptor type 3 (S1PR3) uncoupled from G protein in HeLa cells. Further studies indicated that α-Syn treatment reduced cathepsin D activity while enhancing the secretion of immature pro-cathepsin D into cell culture medium, suggesting that lysosomal delivery of cathepsin D was disturbed. Actually, extracellular α-Syn attenuated the retrograde trafficking of insulin-like growth factor-II/mannose 6-phosphate (IGF-II/M6P) receptor, which is under the regulation of S1PR3. These findings shed light on the understanding of dissemination of the PD pathology, that is, the mechanism underlying how extracellular α-Syn secreted from pathogenic cells causes lysosomal dysfunction of the neighboring healthy cells, leading to propagation of the disease.

Features and mechanisms of propofol-induced protein kinase C (PKC) translocation and activation in living cells.

Background and purpose: In this study, we aimed to elucidate the action mechanisms of propofol, particularly those underlying propofol-induced protein kinase C (PKC) translocation. Experimental approach: Various PKCs fused with green fluorescent protein (PKC-GFP) or other GFP-fused proteins were expressed in HeLa cells, and their propofol-induced dynamics were observed using confocal laser scanning microscopy. Propofol-induced PKC activation in cells was estimated using the C kinase activity receptor (CKAR), an indicator of intracellular PKC activation. We also examined PKC translocation using isomers and derivatives of propofol to identify the crucial structural motifs involved in this process. Key results: Propofol persistently translocated PKCα conventional PKCs and PKCδ from novel PKCs (nPKCs) to the plasma membrane (PM). Propofol translocated PKCδ and PKCη of nPKCs to the Golgi apparatus and endoplasmic reticulum, respectively. Propofol also induced the nuclear translocation of PKCζ of atypical PKCs or proteins other than PKCs, such that the protein concentration inside and outside the nucleus became uniform. CKAR analysis revealed that propofol activated PKC in the PM and Golgi apparatus. Moreover, tests using isomers and derivatives of propofol predicted that the structural motifs important for the induction of PKC and nuclear translocation are different. Conclusion and implications: Propofol induced the subtype-specific intracellular translocation of PKCs and activated PKCs. Additionally, propofol induced the nuclear translocation of PKCs and other proteins, probably by altering the permeability of the nuclear envelope. Interestingly, propofol-induced PKC and nuclear translocation may occur via different mechanisms. Our findings provide insights into the action mechanisms of propofol.

Myogenetic Oligodeoxynucleotides as Anti-Nucleolin Aptamers Inhibit the Growth of Embryonal Rhabdomyosarcoma Cells.

Embryonal rhabdomyosarcoma (ERMS) is the muscle-derived tumor retaining myogenic ability. iSN04 and AS1411, which are myogenetic oligodeoxynucleotides (myoDNs) serving as anti-nucleolin aptamers, have been reported to inhibit the proliferation and induce the differentiation of myoblasts. The present study investigated the effects of iSN04 and AS1411 in vitro on the growth of multiple patient-derived ERMS cell lines, ERMS1, KYM1, and RD. RT-PCR and immunostaining revealed that nucleolin was abundantly expressed and localized in nucleoplasm and nucleoli in all ERMS cell lines, similar to myoblasts. Both iSN04 and AS1411 at final concentrations of 10-30 µM significantly decreased the number of all ERMS cells; however, their optimal conditions were different among the cell lines. In all ERMS cell lines, iSN04 at a final concentration of 10 µM markedly reduced the ratio of EdU+ cells, indicating the inhibition of cell proliferation. Quantitative RT-PCR or immunostaining of phosphorylated histone H3 and myosin heavy chain demonstrated that iSN04 suppressed the cell cycle and partially promoted myogenesis but did not induce apoptosis in ERMS cells. Finally, both iSN04 and AS1411 at final concentrations of 10-30 µM disrupted the formation and outgrowth of RD tumorspheres in three-dimensional culture mimicking in vivo tumorigenesis. In conclusion, ERMS cells expressed nucleolin, and their growth was inhibited by the anti-nucleolin aptamers, iSN04 and AS1411, which modulates several cell cycle-related and myogenic gene expression. The present study provides evidence that anti-nucleolin aptamers can be used as nucleic acid drugs for chemotherapy against ERMS.

Influence of ω3 fatty acids on maternal behavior and brain oxytocin in the murine perinatal period.

INTRODUCTION: Perinatal women often experience mood disorders and postpartum depression due to the physical load and the rapid changes in hormone levels caused by pregnancy, childbirth, and nursing. When the mother's emotions become unstable, their parental behavior (maternal behavior) may decline, the child's attachment may weaken, and the formation of mother-child bonding can become hindered. As a result, the growth of the child may be adversely affected. The objective of this study was to investigate the effect of ω3 fatty acid deficiency in the perinatal period on maternal behavior and the oxytocin concentration and fatty acid composition in brain tissue. MATERIALS AND METHODS: Virgin female C57BL/6 J mice fed a ω3 fatty acid-deficient (ω3-Def) or adequate (ω3-Adq) diet were mated for use in this study. To assess maternal behavior, nest shape was evaluated at a fixed time from gestational day (GD) 15 to postpartum day (PD) 13, and a retrieval test was conducted on PD 3. For neurochemical measurement, brains were removed from PD 1-6 dams and hippocampal fatty acids and hypothalamic oxytocin concentrations were assessed. RESULTS: Peripartum nest shape scores were similar to those reported previously (Harauma et al., 2016); nests in the ω3-Def group were small and of poor quality whereas those in the ω3-Adq group were large and elaborate. The inferiority of nest shape in the ω3-Def group continued from PD 0-7. In the retrieval test performed on PD 3, dams in the ω3-Def group took longer on several parameters compared with those in the ω3-Adq group, including time to make contact with pups (sniffing time), time to start retrieving the next pup (interval time), and time to retrieve the last pup to the nest (grouping time). Hypothalamic oxytocin concentrations on PD 1-6 were lower in the ω3-Def group than in the ω3-Adq group. DISCUSSION: Our data show that ω3 fatty acid deficiency reduces maternal behavior, a state that continued during pup rearing. This was supported by the observed decrease in hypothalamic oxytocin concentration in the ω3-Def group. These results suggest that ω3 fatty acid supplementation during the perinatal period is not only effective in delivering ω3 fatty acids to infants but is also necessary to activate high-quality parental behavior in mothers.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

BACKGROUND: Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms. METHODS: Messenger RNA samples extracted from cultured urine-derived cells from a healthy control and a Dent disease-2 patient were examined to detect the 5' end of the OCRL isoform. For protein expression and functional analysis, vectors containing the full-length OCRL transcripts, the isoform transcripts and transcripts with truncating mutations detected in Lowe syndrome and Dent disease-2 patients were transfected into HeLa cells. RESULTS: We successfully cloned the novel isoform transcripts from OCRL exons 6-24, including the translation-initiation codons present in exon 8. In vitro protein-expression analysis detected proteins of two different sizes (105 and 80 kDa) translated from full-length OCRL, whereas only one protein (80 kDa) was found from the isoform and Dent disease-2 variants. No protein expression was observed for the Lowe syndrome variants. The isoform enzyme activity was equivalent to that of full-length OCRL; the Dent disease-2 variants retained >50% enzyme activity, whereas the Lowe syndrome variants retained <20% activity. CONCLUSIONS: We elucidated the molecular mechanism underlying the two different phenotypes in OCRL-related diseases; the functional OCRL isoform translated starting at exon 8 was associated with this mechanism.

Ethnic comparison in takotsubo syndrome: novel insights from the International Takotsubo Registry.

BACKGROUND: Ethnic disparities have been reported in cardiovascular disease. However, ethnic disparities in takotsubo syndrome (TTS) remain elusive. This study assessed differences in clinical characteristics between Japanese and European TTS patients and determined the impact of ethnicity on in-hospital outcomes. METHODS: TTS patients in Japan were enrolled from 10 hospitals and TTS patients in Europe were enrolled from 32 hospitals participating in the International Takotsubo Registry. Clinical characteristics and in-hospital outcomes were compared between Japanese and European patients. RESULTS: A total of 503 Japanese and 1670 European patients were included. Japanese patients were older (72.6 ± 11.4 years vs. 68.0 ± 12.0 years; p < 0.001) and more likely to be male (18.5 vs. 8.4%; p < 0.001) than European TTS patients. Physical triggering factors were more common (45.5 vs. 32.0%; p < 0.001), and emotional triggers less common (17.5 vs. 31.5%; p < 0.001), in Japanese patients than in European patients. Japanese patients were more likely to experience cardiogenic shock during the acute phase (15.5 vs. 9.0%; p < 0.001) and had a higher in-hospital mortality (8.2 vs. 3.2%; p < 0.001). However, ethnicity itself did not appear to have an impact on in-hospital mortality. Machine learning approach revealed that the presence of physical stressors was the most important prognostic factor in both Japanese and European TTS patients. CONCLUSION: Differences in clinical characteristics and in-hospital outcomes between Japanese and European TTS patients exist. Ethnicity does not impact the outcome in TTS patients. The worse in-hospital outcome in Japanese patients, is mainly driven by the higher prevalence of physical triggers. TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique Identifier: NCT01947621.

Constitutive activation of S1P receptors at the trans-Golgi network is required for surface transport carrier formation.

The importance of the G-protein ßγ subunits in the regulation of cargo transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is well accepted; however, the molecular mechanism underlying the G-protein activation at the TGN remains unclear. We show here that sphingosine 1-phosphate (S1P) receptors at the PM were trafficked to the TGN in response to a surface transport cargo, temperature-sensitive vesicular stomatitis virus glycoprotein tagged with green fluorescent protein accumulation in the Golgi. The receptor internalization occurred in an S1P-independent manner but required phosphorylation by G-protein receptor kinase 2 and ß-arrestin association before internalization. Continuously activated S1P receptors in a manner dependent on S1P at the TGN kept transmitting G-protein signals including the ßγ subunits supply necessary for transport carrier formation at the TGN destined for the PM.

Myogenetic Oligodeoxynucleotide (myoDN) Recovers the Differentiation of Skeletal Muscle Myoblasts Deteriorated by Diabetes Mellitus.

Skeletal muscle wasting in patients with diabetes mellitus (DM) is a complication of decreased muscle mass and strength, and is a serious risk factor that may result in mortality. Deteriorated differentiation of muscle precursor cells, called myoblasts, in DM patients is considered to be one of the causes of muscle wasting. We recently developed myogenetic oligodeoxynucleotides (myoDNs), which are 18-base single-strand DNAs that promote myoblast differentiation by targeting nucleolin. Herein, we report the applicability of a myoDN, iSN04, to myoblasts isolated from patients with type 1 and type 2 DM. Myogenesis of DM myoblasts was exacerbated concordantly with a delayed shift of myogenic transcription and induction of interleukins. Analogous phenotypes were reproduced in healthy myoblasts cultured with excessive glucose or palmitic acid, mimicking hyperglycemia or hyperlipidemia. iSN04 treatment recovered the deteriorated differentiation of plural DM myoblasts by downregulating myostatin and interleukin-8 (IL-8). iSN04 also ameliorated the impaired myogenic differentiation induced by glucose or palmitic acid. These results demonstrate that myoDNs can directly facilitate myoblast differentiation in DM patients, making them novel candidates for nucleic acid drugs to treat muscle wasting in patients with DM.

Blood flow distribution after end-to-side anastomosis with wide arteriotomy in extremity free flap surgery.

PURPOSE: Although many studies have investigated the optimal anastomotic procedure for the end-to-side (ETS) procedure with a free flap, no study has focused on the size of the arteriotomy. Some surgeons have recently described the effectiveness of ETS with wide arteriotomy, but the postoperative haemodynamics remains unclear for free flaps created using this technique. The aim of this study was to use ultrasonography to evaluate the postoperative blood flow distribution after ETS with a wide arteriotomy in extremity free flap surgery. METHODS: We evaluated 20 free flaps in 18 consecutive patients who received an ultrasonographic examination after free flap surgery using the ETS technique with wide arteriotomy for arterial anastomosis. All flaps were examined after surgery and blood flow was calculated for the flap and recipient vessels. RESULTS: All 20 flaps survived, but one flap developed asymptomatic arterial thrombosis and 19 flaps were analysed. For the ETS technique with wide arteriotomy, peripheral circulation was well preserved in all flaps. Comparison of flap types showed that blood flow was significantly higher in myocutaneous flaps than in fasciocutaneous flaps, but there was no significant difference according to the size of the arteriotomy. CONCLUSIONS: Given the range of arteriotomy performed using the ETS with a wide arteriotomy technique, the blood flow volume in the flap depended on the type of flap but not on the size of the arteriotomy. A steal phenomenon related to the creation of a wide window in the receipt artery was not found in the analysed retrospective cohort.

Rapid Lipid Modification of Endothelial Cell Membranes in Cardiac Ischemia/Reperfusion Injury: a Novel Therapeutic Strategy to Reduce Infarct Size.

PURPOSE: Plasma membranes constitute a gathering point for lipids and signaling proteins. Lipids are known to regulate the location and activity of signaling proteins under physiological and pathophysiological conditions. Membrane lipid therapies (MLTs) that gradually modify lipid content of plasma membranes have been developed to treat chronic disease; however, no MLTs have been developed to treat acute conditions such as reperfusion injury following myocardial infarction (MI) and percutaneous coronary intervention (PCI). A fusogenic nanoliposome (FNL) that rapidly incorporates exogenous unsaturated lipids into endothelial cell (EC) membranes was developed to attenuate reperfusion-induced protein signaling. We hypothesized that administration of intracoronary (IC) FNL-MLT interferes with EC membrane protein signaling, leading to reduced microvascular dysfunction and infarct size (IS). METHODS: Using a myocardial ischemia/reperfusion swine model, the efficacy of FNL-MLT in reducing IS following a 60-min coronary artery occlusion was tested. Animals were randomized to receive IC Ringer's lactate solution with or without 10 mg/mL/min of FNLs for 10 min prior to reperfusion (n = 6 per group). RESULTS: The IC FNL-MLT reduced IS (25.45 ± 16.4% vs. 49.7 ± 14.1%, P < 0.02) and enhanced regional myocardial blood flow (RMBF) in the ischemic zone at 15 min of reperfusion (2.13 ± 1.48 mL/min/g vs. 0.70 ± 0.43 mL/min/g, P < 0.001). The total cumulative plasma levels of the cardiac injury biomarker cardiac troponin I (cTnI) were trending downward but were not significant (999.3 ± 38.7 ng/mL vs. 1456.5 ± 64.8 ng/mL, P = 0.1867). However, plasma levels of heart-specific fatty acid binding protein (hFABP), another injury biomarker, were reduced at 2 h of reperfusion (70.3 ± 38.0 ng/mL vs. 137.3 ± 58.2 ng/mL, P = 0.0115).  CONCLUSION: The IC FNL-MLT reduced IS compared to vehicle in this swine model. The FNL-MLT maybe a promising adjuvant to PCI in the treatment of acute MI.

Differences in pharmacological property between combined therapy of the vasopressin V2-receptor antagonist tolvaptan plus furosemide and monotherapy of furosemide in patients with hospitalized heart failure.

BACKGROUND: Tolvaptan has been shown to improve congestion in heart failure patients. The purpose of this study was to evaluate the pharmacology and clinical efficacy of combined tolvaptan and furosemide therapy. METHODS: This study included 40 patients with systemic volume overload who were hospitalized for heart failure. Patients who showed no improvement in the condition after receiving 20 mg intravenous furosemide were included and were randomly selected to receive tolvaptan as an add-on to furosemide or to receive an increased dose of furosemide. We evaluated the bioelectrical impedance analyzer parameters, the parameters of the inferior vena cava using echocardiography, vital signs, body weight, urine output, and laboratory data for 5 days. RESULTS: In the changes from baseline between intracellular water volume (ICW) and extracellular water volume (ECW) after additional use of tolvaptan or furosemide from Day 1 to Day 5, there were no significant differences observed between ICW and ECW over 5 days in the tolvaptan + furosemide group, although differences were found in the furosemide group from Day 2 onward. Changes in the respiratory collapse of inferior vena cava increased significantly, and systolic blood pressure decreased significantly only in the furosemide group. CONCLUSIONS: The present study clearly demonstrates that combined therapy with tolvaptan and furosemide removed excess ICW and ECW to an equal extent, while furosemide alone primarily removed ECW, including intravascular water.

Clinical significance of arterial stiffness as a factor for hospitalization of heart failure with preserved left ventricular ejection fraction: a retrospective matched case-control study.

BACKGROUND: Previous studies have been conducted to identify characteristics of patients with heart failure with preserved ejection fraction (HFpEF), but the risk factors of HFpEF remain unclear. We investigated the associations between arterial stiffness and the risk of hospitalization for HFpEF patients. METHODS: For the case group, we enrolled patients with preserved EF who had been hospitalized for HF from April 2013 to March 2015 and examined the cardio-ankle vascular index (CAVI). For the control group, we enrolled outpatients with preserved EF and with hypertension, diabetes mellitus, dyslipidemia, and/or coronary artery disease but who did not present with HF symptoms and had never been diagnosed or treated for HF during the same period. The control group matched with the case group for age and sex. The association between hospitalized HFpEF and clinical variables was analyzed using conditional logistic regression models. RESULTS: The CAVI value was significantly higher in patients with hospitalized HFpEF compared with patients with the control [10.4 (9.8-11.0) vs. 9.2 (8.1-10.0), p < 0.001). On the multivariate conditional logistic regression analysis, high CAVI (OR 6.76, 95% CI 2.28-20.10, p < 0.001) and anemia (OR 3.91, 95% CI 1.47-10.40, p = 0.006) were independently associated with hospitalization of HFpEF patients. CONCLUSIONS: The present study has demonstrated that the high value of CAVI was independently associated with the hospitalization of HFpEF patients.

Berberine and palmatine inhibit the growth of human rhabdomyosarcoma cells.

A natural isoquinoline alkaloid, berberine, has been known to exhibit anti-tumor activity in various cancer cells via inducing cell cycle arrest. However, it has not been investigated whether berberine and its analogs inhibit the growth of rhabdomyosarcoma (RMS), which is the most frequent soft tissue tumor in children. The present study examined the anti-tumor effects of berberine and palmatine on expansions of three human embryonal RMS cell lines; ERMS1, KYM1, and RD. Intracellular incorporation of berberine was relatively higher than that of palmatine in every RMS cell line. Berberine significantly inhibited the cell cycle of all RMS cells at G1 phase. On the other hand, palmatine only suppressed the growth of RD cells. Both of berberine and palmatine strongly inhibited the growth of tumorsphere of RD cells in three-dimensional culture. These results indicate that berberine derivatives have the potential of anti-tumor drugs for RMS therapy.Abbreviations: ARMS: alveolar rhabdomyosarcoma; ERMS: embryonal rhabdomyosarcoma; RMS: rhabdomyosarcoma.

Involvement of Receptor-Mediated S1P Signaling in EGF-Induced Macropinocytosis in COS7 Cells.

Macropinocytosis is a highly conserved cellular process of endocytosis by which extracellular fluid and nutrients are taken up into cells through large, heterogeneous vesicles known as macropinosomes. Growth factors such as epidermal growth factor (EGF) can induce macropinocytosis in many types of cells, although precise mechanism underlying EGF-induced macropinocytosis remains unclear. In the present studies we have shown the involvement of S1P signaling in EGF-induced macropinocytosis in COS7 cells. First, EGF-induced macropinocytosis was strongly impaired in sphingosine kinase isozymes, SphK1 or SphK2-depleted cells, which was completely rescued by the expression of the corresponding wild-type isozyme but not the catalytically inactive one, suggesting the involvement of sphingosine 1-phosphate (S1P) in this phenomenon. Next, we observed that EGF-induced macropinocytosis was strongly inhibited in S1P type 1 receptor (S1P1R)-knockdown cells, implying involvement of S1P1R in this event. Furthermore, we could successfully demonstrate EGF-induced trans-activation of S1P1R using one-molecular fluorescence resonance energy transfer (FRET) technique. Moreover, for EGF-induced Rac1 activation, a step essential to F-actin formation and subsequent macropinocytosis, S1P signaling is required for its full activation, as judged by FRET analysis. These findings indicate that growth factors such as EGF utilize receptor-mediated S1P signaling for the regulation of macropinocytosis to fulfil vital cell activity.

Identification of the Myogenetic Oligodeoxynucleotides (myoDNs) That Promote Differentiation of Skeletal Muscle Myoblasts by Targeting Nucleolin.

Herein we report that the 18-base telomeric oligodeoxynucleotides (ODNs) designed from the Lactobacillus rhamnosus GG genome promote differentiation of skeletal muscle myoblasts which are myogenic precursor cells. We termed these myogenetic ODNs (myoDNs). The activity of one of the myoDNs, iSN04, was independent of Toll-like receptors, but dependent on its conformational state. Molecular simulation and iSN04 mutants revealed stacking of the 13-15th guanines as a core structure for iSN04. The alkaloid berberine bound to the guanine stack and enhanced iSN04 activity, probably by stabilizing and optimizing iSN04 conformation. We further identified nucleolin as an iSN04-binding protein. Results showed that iSN04 antagonizes nucleolin, increases the levels of p53 protein translationally suppressed by nucleolin, and eventually induces myotube formation by modulating the expression of genes involved in myogenic differentiation and cell cycle arrest. This study shows that bacterial-derived myoDNs serve as aptamers and are potential nucleic acid drugs directly targeting myoblasts.

Activation of atypical protein kinase C by sphingosine 1-phosphate revealed by an aPKC-specific activity reporter.

Atypical protein kinase C (aPKC) isozymes are unique in the PKC superfamily in that they are not regulated by the lipid second messenger diacylglycerol, which has led to speculation about whether a different second messenger acutely controls their function. Here, using a genetically encoded reporter that we designed, aPKC-specific C kinase activity reporter (aCKAR), we found that the lipid mediator sphingosine 1-phosphate (S1P) promoted the cellular activity of aPKC. Intracellular S1P directly bound to the purified kinase domain of aPKC and relieved autoinhibitory constraints, thereby activating the kinase. In silico studies identified potential binding sites on the kinase domain, one of which was validated biochemically. In HeLa cells, S1P-dependent activation of aPKC suppressed apoptosis. Together, our findings identify a previously undescribed molecular mechanism of aPKC regulation, a molecular target for S1P in cell survival regulation, and a tool to further explore the biochemical and biological functions of aPKC.

Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells.

Sphingosine 1-phosphate (S1P) is a bioactive phosphorylated product of sphingosine catalyzed by sphingosine kinase (SphK) and implicated in diverse cellular functions including vesicular trafficking. In the present study we have shown the importance of one of the subtypes of SphK, SphK2, in the regulation of cargo content in exosomes released from human myeloid leukemia K562 cells. First, SphK2 has been shown to localize with N-Rh-PE-positive late endosomes in the cells. Next, siRNA-mediated knockdown of Sphk2 but not SphK1 resulted in a reduction of cargo content in purified exosomes. The involvement of SphK2 in this phenomenon was further investigated by pharmacological approaches. When cells were treated with N,N-dimethylsphingosine (DMS), one of the most frequently used inhibitors for SphK, cargo contents in purified exosomes were enhanced unexpectedly. Finally, it has been shown that DMS has a potency to stimulate SphK2 activity depending on the substrate sphingosine- and the inhibitor-doses as estimated by in vitro assay systems using a purified SphK2. These findings suggest that SphK2/S1P signaling plays an important role in the regulation of cargo content in exosomes in K562 cells.

Extracellular α-synuclein drives sphingosine 1-phosphate receptor subtype 1 out of lipid rafts, leading to impaired inhibitory G-protein signaling.

α-Synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson's disease (PD). Although growing evidence suggests that cell-to-cell transmission of α-Syn is associated with the progression of PD and that extracellular α-Syn promotes formation of inclusion bodies, its precise mechanism of action in the extracellular space remains unclear. Here, as indicated by both conventional fractionation techniques and FRET-based protein-protein interaction analysis, we demonstrate that extracellular α-Syn causes expulsion of sphingosine 1-phosphate receptor subtype 1 (S1P1R) from the lipid raft fractions. S1P1R regulates vesicular trafficking, and its expulsion involved α-Syn binding to membrane-surface gangliosides. Consequently, the S1P1R became refractory to S1P stimulation required for activating inhibitory G-protein (Gi) in the plasma membranes. Moreover, the extracellular α-Syn also induced uncoupling of the S1P1R on internal vesicles, resulting in the reduced amount of CD63 molecule (CD63) in the lumen of multivesicular endosomes, together with a decrease in CD63 in the released exosomes from α-Syn-treated cells. Furthermore, cholesterol-depleting agent-induced S1P1R expulsion from the rafts also resulted in S1P1R uncoupling. Taken together, these results suggest that extracellular α-Syn-induced expulsion of S1P1R from lipid rafts promotes the uncoupling of S1P1R from Gi, thereby blocking subsequent Gi signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes. These findings help shed additional light on PD pathogenesis.

Erratum: Impairment of PDGF-induced chemotaxis by extracellular α-synuclein through selective inhibition of Rac1 activation.

This corrects the article DOI: 10.1038/srep37810.