Inhibition of acid sphingomyelinase disrupts LYNUS signaling and triggers autophagy

• DOI: 10.1194/jlr.m080242
• Publication Date: 2018-01-29T16:05:55Z
• Authors: Matthew J. Justice; Irina Bronova; Kelly S. Schweitzer; Christophe Poirier; Janice S. Blum; Evgeny V. Berdyshev; Irina Petrache
• Keywords: Acid sphingomyelinase

Activation of the lysosomal ceramide-producing enzyme, acid sphingomyelinase (ASM), by various stresses is centrally involved in cell death and has been implicated autophagy. We set out to investigate role baseline ASM activity maintaining physiological functions lysosomes, focusing on nutrient-sensing complex (LYNUS), a membrane-anchored multiprotein that includes mammalian target rapamycin (mTOR) transcription factor EB (TFEB). inhibition with imipramine or sphingomyelin phosphodiesterase 1 (SMPD1) siRNA human lung cells, transgenic Smpd1+/− haploinsufficiency mouse lungs, markedly reduced mTOR- P70-S6 kinase (Thr 389)-phosphorylation modified TFEB pattern consistent its activation. Inhibition significantly increased autophagy preserved degradative potential. Pulse labeling sphingolipid metabolites revealed decreased sphingosine (Sph) Sph-1-phosphate (S1P) levels at level ceramide hydrolysis. These findings suggest maintain mTOR signaling inhibit implicate Sph and/or S1P control function. The activation above homeostatic typically apoptosis via production ceramide, recently stress-induced macro-autophagy (referred as autophagy, henceforth) (1.Patschan S. Chen J. Polotskaia A. Mendelev N. Cheng Patschan D. Goligorsky M.S. Lipid mediators premature senescence endothelial cells.Am. Physiol. Heart Circ. 2008; 294: H1119-H1129Crossref PubMed Scopus (95) Google Scholar, 2.Lee J.K. Jin H.K. Park M.H. Kim B.R. Lee P.H. Nakauchi H. Carter J.E. He X. Schuchman E.H. Bae J.S. Acid modulates autophagic process controlling biogenesis Alzheimer's disease.J. Exp. Med. 2014; 211: 1551-1570Crossref (106) Scholar). Despite understanding involvement ASM's brisk stress response, little known about function during conditions. causal link between loss lipid storage disorder, Niemann-Pick disease, requirement for proper cellular highlighted essential breakdown membrane homeostasis (3.Schuchman sphingomyelinase, membranes disease: lessons from disease.FEBS Lett. 2010; 584: 1895-1900Crossref Docked membrane, (LYNUS) controls protein synthesis an mechanism survival starvation (4.Settembre C. Fraldi Medina D.L. Ballabio Signals lysosome: centre clearance energy metabolism.Nat. Rev. Mol. Cell Biol. 2013; 14: 283-296Crossref (1081) In turn, aberrant pathogenesis several diseases, including chronic obstructive pulmonary diseases (5.Chen Z.H. H.P. Sciurba F.C. S.J. Feghali-Bostwick Stolz D.B. Dhir R. Landreneau R.J. Schuchert M.J. Yousem S.A. et al.Egr-1 regulates cigarette smoke-induced disease.PLoS One. 3: e3316Crossref (371) Therefore, molecular determinants clinical importance. secreted forms ASM, both being encoded gene, catalyze, optimally acidic pH, hydrolysis into phosphorylcholine. rapidly stress, generating excess that, interacts specific molecules trigger second messenger effects. addition, incorporates alters biophysical properties bilayers, such fluidity stiffness, dislodging cholesterol rafts, impacting acyl chain order parameters neighboring lipids, modifying bilayer composition (6.Yu Alterman M. Dobrowsky R.T. Ceramide displaces rafts decreases association binding caveolin-1.J. Res. 2005; 46: 1678-1691Abstract Full Text PDF (104) Because ceramides produced lysosome are not shuttled other subcellular compartments, they may directly influence coalescence microdomains required (7.Chatelut Leruth Harzer K. Dagan Marchesini Gatt Salvayre Courtoy P. Levade T. Natural unable escape lysosome, contrast fluorescent analogue.FEBS 1998; 426: 102-106Crossref (68) However, ceramidase, which metabolizes (Sph), molecule is, itself, alterations calcium efflux (8.Höglinger Haberkant Aguilera-Romero Riezman Porter F.D. Platt F.M. Galione Schultz Intracellular releases lysosomes.eLife. 2015; 4: e10616Crossref (89) investigated determine whether LYNUS conditions, hypothesizing sufficient associated disrupted signaling. During plentitude ATP, amino acids, growth signals, (mTOR), serine/threonine component LYNUS, stimulates This fundamental requires docking ensured farnesylated component, Ras homolog enriched brain (RHEB) Upon becomes inactive one phosphorylation targets (TFEB), translocates nucleus, where it upregulates genes own (9.Settembre De Cegli Mansueto G. Saha P.K. Vetrini F. Visvikis O. Huynh Carissimo Palmer Klisch T.J. al.TFEB metabolism through starvation-induced autoregulatory loop.Nat. 15: 647-658Crossref (630) When activated levels, triggers impaired degradation (decreased flux) coupled mRNA transcripts (2.Lee Whether this effect due perturbations remains unclear. Such sphingolipids either have effects, e.g., engages similar induced depletion (10.Guenther G.G. Peralta E.R. Rosales K.R. Wong S.Y. Siskind L.J. Edinger A.L. starves cells downregulating nutrient transporter proteins.Proc. Natl. Acad. Sci. USA. 105: 17402-17407Crossref (145) Scholar), could alter fluidity, potentially affecting anchoring. Utilizing primary epithelial murine we show inactivate components initiate potential marked reductions Sph, rather than levels. Unless otherwise stated, all reagents were purchased Sigma-Aldrich. All experiments performed full (containing supplements) medium unless stated. Primary artery (HPAECs) (Invitrogen, C0085C) cultured Medium 200 M200500) low serum supplement S00310). microvascular (HLMVECs) CC2527) EGM-2MV BulletKit CC3202). umbilical vein (HUVECs), kind gift Dr. Mathias Clauss (Indiana University), VascuLife VEGF complete kit (LifeLine, LL0003). THP-1 monocytes (ATCC, TIB202) RPMI 11875119) supplemented 10% FBS (Thermo Fisher Scientific, sh30910-03HI). Immortalized bronchial (BEAS2b cells) DMEM high glucose pyruvate 11995-073), 1% penicillin/streptomycin (Sigma, P4333-100ML). incubated 37°C 5% CO2 100% humidity. 80–90% confluent, was replaced vehicle added indicated time, when washed 5 ml PBS 4°C scraped 1.5 4°C. Cells then spun 16,000 g 10 min pellets snap-frozen liquid nitrogen until assayed. Lysosomal measured Amplex Red A12220) radioactivity. Briefly, utilizes indirect two-step reaction produces readout activity. ice-cold end treatment, PBS, centrifuged min. Supernatant removed, remaining pellet nitrogen. At time assay, hydrated buffer activity, mM sphingomyelin, 2 U/ml HRP, 8 alkaline phosphatase. Kinetic fluorescence measurements read using wavelength 590 nm over period h SpectraMax m2e microplate reader SoftMax Pro software. For radioactive lysed 75 μl containing 25 Tris (pH 7.6), EDTA, 0.2% Triton-X, phosphatase inhibitors, protease inhibitors. To 4 same used lyse 3.08 14C-choline methyl 55 mCi/mmol 0.1 mCi/ml create substrate (1.4 pmol/μl). 103 buffer, 35 lysate, 12 0.2 M acetic acid, 50 added, mixed vortex, allowed react 2.3 h. stop reaction, 250 2:1 (chloroform:methanol) solution vortexed. this, 800 (methanol:chloroform) plus water From aqueous phase, extracted MicroScint PS (Perkin-Elmer, 6013631) radioactivity quantified Topcount scintillation counter. once gently Then, supernatant removed thawed lysis Triton-X (EMD, 9002931), 150 NaCl Fisher, BP3581), 7.6; Invitrogen, 15504020) vigorously vortexed five times period, followed centrifugation use assays whole lysate. Protein concentration determined BCA (Pierce, 23227). Equal amounts (2–20 μg) diluted Laemmli 4× (reducing) (Boston Bio Products, NC9099736) resolved Criterion 12+2-well 4–20% TGX gels (Bio-Rad, 5671093). A semi-dry transfer apparatus 1703848) employed proteins polyvinylidene fluoride IPVH00010). Membranes probed following antibodies: anti-β-actin (A5441), anti-microtubule-associated light 3β (LC3B) L7543), anti-vinculin (Abcam, ab10858), anti-GAPDH ab9485), anti-phospho (P70-S6k) 389) (Cell Signaling Technologies, 9205), (Ser 2448) (p-mTOR) 5536). Appropriate secondary antibodies (goat anti-rabbit/mouse, HRP conjugate) (GE Healthcare, 45001175/45001187) conjunction ECL Prime RPN2232) Luminata Forte (EMDMillipore, WBLUF0500) chemiluminescent reaction. Images taken ChemiDoc (Bio-Rad) XRS system ImageLab Density quantification bands Western blots ImageJ software (The National Institutes Health, Bethesda, MD; http://imagej.nih.gov/ij/). Quantification interest relative intensity respective loading ratio equal experimental (e.g., vehicle-treated untreated) group. Nucleofector kits (Lonza, VVPI-1001) Amaxa transfect HPAECs siGenome (Dharmacon, siGlo/non-targeting pool 1/non-targeting 2/SMPD1, D0016300205/D-001206-13/D-001206-14/M-006676-01-0020) U-017 program per manufacturer's protocol. listed achieved inside cuvette prior electroporation. After electroporation, tissue culture dishes medium. changed 18 24 after harvested 48 72 transfection, indicated. speed min, RNA GenElute total mini RTN70-1KT) protocol, cDNA reverse transcribed capacity RNase inhibitor (Applied Biosystems, 4374966). Using SYBR Select Master Mix 4472908) SMPD1/PGK1 primers (Qiagen, PPH02494A/PPH02049A), ng amplified ABI 7500 real-time PCR Biosystems). (125,000 well) mm glass coverslips (VWR, 48380046) residing 12-well (Costar, 3513) coated gelatin-based coating Biologics, 6950) were, treatment fixation formaldehyde, exposed rabbit anti-TFEB polyclonal antibody, Alexa Fluor 555 conjugated (Bioss, bs-5137R-A555), mounted SlowFade Gold Antifade reagent DAPI S36939) microscope slides 12-544-3). Nikon Eclipse 80i microscope. BEAS2b stably express eGFP-LC3B 4-well Lab-Tek 154526) (50,000 well). lysosome-associated (LAMP1) anti-LAMP1 Technology, 9091), 594 A20185), 12-544-14) imaged washing (Fisher), fixed glutaraldehyde G7776) formaldehyde 28908), 2% phosphate 30 room temperature. two rounds further processed Electron Microscopy Core Facility, Indiana University School Medicine, included post fixing, embedding, cutting, mounting slides. Tecnai G2 Twin (FEI, Hillsboro) equipped AMR CCD (Advanced Techniques). cotransfected (Amaxa U-017) expression plasmid (pEF6/V5-His) expressing C-terminally tagged (11.Zeidan Y.H. Hannun Y.A. Cdelta-mediated phosphorylation.J. Chem. 2007; 282: 11549-11561Abstract (123) Yusuf (Stony Brook University) N-terminally under cytomegalovirus promoter (12.Jackson W.T. Giddings Jr., T.H. Taylor M.P. Mulinyawe Rabinovitch Kopito R.R. Kirkegaard Subversion autophagosomal machinery viruses.PLoS e156Crossref (681) Scholar) (a William Jackson, Stanford University). verify punctate formation stimuli, transiently transfected treated chloroquine (supplemental Fig. S1A). Selection pressure applied Geneticin (Santa Cruz, sc29065) passages. passaged overexpression confirmed immunoblot S1). confluent indicated, removal Accutase (10 ml) addition dishes. centrifuges kept procedures. collected pipetting 500 Pellets suspended again removing supernatants, B Axis-Shield Application S53 An aliquot immediately prepare samples. each samples generated homogenized separately (30 strokes) Dounce homogenizer pestle B. Samples labeled cytosolic fraction organelle fraction. resuspended C, S53. 14 gradient discontinuous steps made v/v dilutions OptiPrep follows: (sample), 12%, 14%, 16%, 18%, 20%, 25%, 150,000 20 Fractions bottom 3 syringe inch 16 gauge blunt needle. Six fractions collected, μl, starting six Bioproducts, blotting decanted methanol extraction. Methanol, water, acetonitrile (HPLC grade) Burdick Jackson (Muskegon, MI). dihydro (DH)Sph, 17-carbon analog (C17-Sph), S1P, DHS1P, (C17-S1P), N-acylated (C14:0, C16:0, C18:1, C18:0, C20:0, C24:1, C24:0) Sphs (Ceramides, Cer), N-heptadecanoyl-Sph (C17:0-Cer), sphingosylphosphorylcholines (sphingomyelins, N-12:0, N-16:0, N-18:0, N-24:1, N-24:0), glucosylceramides (N-16:0, N-24:0) obtained Avanti Polar Lipids (Alabaster, AL). N-D3-16:0-glucosylceramide N-D3-16:0-lactosylceramide Matreya LLC (State College, PA). standards dissolved (sphingoid base phosphates trace amount concentrated hydrochloric acid) stored −20°C. Pyridine (acetylation anhydride products Alltech Associates (Deerfield, IL). Cellular lipids Bligh Dyer procedure N HCl phase separation (13.Berdyshev E.V. Gorshkova I.A. Usatyuk Zhao Y. Saatian Hubbard W. Natarajan V. novo biosynthesis dihydrosphingosine-1-phosphate cells.Cell. Signal. 2006; 18: 1779-1792Crossref (75) C17-S1P pmols), C17-Sph N-C17:0-Cer N-12:0-sphingosylphosphorylcholine (12:0-SM, 100 (25 pmol), internal standards, initial step ethanol aliquots phospholipid content, described stream nitrogen, transferred autosampler vials, subjected LC-MS/MS. (for h, indicated) EBM-2 L-[U-13C,15N]serine (50 mg/l; Cambridge Isotope Laboratories, Andover, MA). Imipramine beginning labeling, respectively. incubation, above. Quantitation label-incorporated LC-MS/MS monitoring [M+3] isotopes precursor product ions, below. Analyses sphingoid base-1-phosphates, ceramides, bases, sphingomyelins, glycosylceramides, lactosylceramides ESI-LC/MS/MS. instrumentation Sciex 6500 QTRAP hybrid triple quadrupole linear ion-trap mass spectrometer (AB Sciex, Redwood City, CA) Ion Drive Turbo V ionspray ionization source interfaced Shimadzu Nexera X2 UHPLC system. their derivatives separated Ascentis Express RP-Amide 2.7 μm 2.1 × column elution methanol:water:formic (65:35:0.5, ammonium formate) methanol:chloroform:water:formic (90:10:0.5:0.5, formate). DHS1P analyzed bis-acetylated standard employing negative ion ESI multiple (MRM) analysis, (14.Berdyshev Garcia J.G. W.C. Quantitative analysis base-1-phosphates bisacetylated chromatography-tandem spectrometry.Anal. Biochem. 339: 129-136Crossref (112) Ceramides bases C17:0-Cer positive MRM facilitate avoid stable isotope overlap phosphatidylcholines hydrolyzed methylamine 55°C (15.Clarke N.G. Dawson R.M. Alkaline O leads N-transacylation. new method quantitative deacylation phospholipids.Biochem. 1981; 195: 301-306Crossref (186) Reagents evaporated stream. residual nonsaponified sphingomyelins. Sphingomyelins detected ions mode transition corresponding m/z 184 (phosphocholine). Sphingomyelin creating curves variable (N-16:0-, 18:0-, 24:1-, 24:0-sphingomyelins) versus N-12:0-sphingomyelin (internal standard). linearity correlation coefficients regression analysis. species there no available best approximation analogs most structure. approved IACUC Indianapolis, IN) conformed Public Health Service policy humane care laboratory animals outlined Institute Laboratory Animal Research guide animals. Three-month-old female Smpd1+/+, Smpd1+/−, Smpd1−/− mice (C57Bl/6 background strain), Edward Erich Gulbins, harvest perfused Lung blotting. Homogenized sonicated s 30% power, speed, statistical analyses GraphPad Prism (GraphPad Software, La Jolla, CA). First, fluorometric inhibitory μM, h) S2), tertiary amine tricyclic antidepressant increase proteolytic (16.Hurwitz Ferlinz Sandhoff desipramine causes fibroblasts.Biol. Hoppe Seyler. 1994; 375: 447-450Crossref (212) Pharmacological status P70-S6k (within sustainably up h), visualized blot densitometry (Fig. 1A–C). also S3A), but did affect Akt S3B). nonspecific corroborated these results targeting transcript gene (1–3.33 48–72 h). Compared three nontargeting pools fluorescently (siGLO), SMPD1 ∼50%, (q)PCR 1D) inhibited fluorimetrically (by ∼85%, 1E) assay ∼65%, data shown). knockdown P70-S6k, 1F, G). next interrogated TFEB, docked dephosphorylation translocation nucleus. "smear" appearance immunoblotted seen lower weight more band, indicating 1H). apparent nuclear immunostaining, epifluorescence 1I). Together, downstream dephosphorylation, nucleus indicate inhibits normal conditions only Autophagosome abundance first intracellular LC3B-II. (50–100 μM 2–24 LC3B-II 2A–D). Similarly, led significant increases LC3B-II,