The FASEB Journal article fj.14-268474. Published online February 12, 2015.
The FASEB Journal • Research Communication
Role of p38 mitogen-activated protein kinase in linking stearoyl-CoA desaturase-1 activity with endoplasmic reticulum homeostasis Andreas Koeberle,*,1 Carlo Pergola,* Hideo Shindou,† Solveigh C. Koeberle,‡ Takao Shimizu,†,§ Stefan A. Laufer,{ and Oliver Werz* *Institute of Pharmacy, Friedrich-Schiller-University Jena, Jena, Germany; †Department of Lipid Signaling, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan; ‡Leibniz Institute of Age Research, Fritz-Lipmann-Institute, Jena, Germany; §Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; and {Department of Pharmaceutical Chemistry, Pharmaceutical Institute, University of Tubingen, ¨ Tubingen, ¨ Germany Endoplasmic reticulum (ER) homeostasis is regulated by a network of signaling pathways to which stearoyl-CoA desaturase (SCD)-1, p38 mitogen-activated protein kinase (MAPK) and the unfolded protein response (UPR) belong. Because all these pathways are located at the interface of cell cycle control and cell stress, we hypothesized a cross-regulation. Interference with SCD-1, either by small interfering (si)RNA or the specific SCD-1 inhibitor CAY10566 (EC50 1 mM; ‡ 24 h), specifically induced phosphorylation and thus activation of p38 MAPK in NIH-3T3 mouse fibroblasts (1.5- to 2-fold; 48 hours). During lipotoxic and cell cycle stress, prolonged activation of p38 MAPK due to SCD-1 inhibition induced ER stress, the UPR, and ER/Golgi remodeling as shown by Western blot and immunofluorescence microscopy (1.2- to 3.5fold). Specific inhibition of p38 MAPK by Skepinone-L [half maximal inhibitory concentration (IC50) 25–50 nM] reversed these effects (at 1 mM; 48 hours). The specificity by which SCD-1 modulates the phospholipid composition and inhibits p38 MAPK signaling (among survival/stress pathways), thereby preventing ER stress (but not other SCD-1-dependent responses), suggests selective proteinlipid interactions. Palmitoleoyl/oleoyl-phosphatidylinositol (PI) was accordingly identified as potential lipid mediator using chromatography-coupled ESI tandem mass spectrometry. We conclude that the negative regulation of p38 MAPK mediates the protective effects of SCD-1 on ER homeostasis under distinct stress conditions.—Koeberle, A., Pergola, C., Shindou, H., Koeberle, S. C., Shimizu, T., Laufer, S. A., Werz, O. Role of p38 mitogen-activated protein kinase in linking stearoyl-CoA desaturase-1 activity with endoplasmic reticulum homeostasis. FASEB J. 29, 000–000 (2015). www.fasebj.org ABSTRACT
Abbreviations: ATF, activating transcription factor; BiP, binding protein/GRP78; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; ERGIC, endoplasmic reticulum/ Golgi intermediate compartment; FCS, fetal calf serum; HSD, honest significant difference; IC50, half maximal inhibitory concentration; MUFA, monounsaturated fatty acid; PERK, RNA-like endoplasmic reticulum kinase; PI, phosphatidylinositol; SCD, stearoyl-CoA desaturase; siRNA, small interfering RNA; UPR, unfolded protein response
0892-6638/15/0029-0001 © FASEB
Key Words: bioactive lipid • fatty acid • lipidomics • phospholipid unfolded protein response
•
SCD IS THE RATE-LIMITING enzyme catalyzing the conversion of saturated fatty acids into cis-D9 monounsaturated fatty acids (MUFAs) (1). The isoenzyme SCD-1 promotes dietinduced obesity, atherosclerosis, and insulin resistance as well as cell proliferation, survival, and tumorigenesis (1). How SCD-1 modulates these processes is not readily understood. Key might be the suppression of prolonged ER stress (and thus the UPR), thereby promoting the development of many diseases, including atherosclerosis, diabetes, and cancer (2, 3). The UPR is a signaling network from the ER to the nucleus that senses ER stress and coordinates homeostatic and apoptotic responses (4). The 3 major branches of the UPR are kept in an inactive state by the chaperone binding protein (BiP/GRP78). Activation of the RNA-like ER kinase (PERK) enhances phosphorylation of eukaryotic initiation factor 2 and induces the expression of activating transcription factor (ATF)4 and C/EBP homologous protein (CHOP), which attenuates protein biosynthesis and induces cell cycle arrest. How SCD-1 activity is related to ER stress and the UPR is largely unknown. It has been suggested that an increase of phospholipid saturation upon SCD-1 inhibition induces the UPR (2) either through disturbing Ca2+ homeostasis (5) or activating PERK (6). However, these signaling routes require dramatic changes in the proportion of saturated phospholipids ($20 mol %) (5, 6), which are neither achieved by deletion nor inhibition of SCD-1 (3–12 mol % depending on cell type and experimental conditions) (2, 7, 8), suggesting alternative mechanisms being involved. The stress-activated kinase p38 MAPK is a key enzyme for survival and inflammation and plays a dual role in the UPR 1 Correspondence: Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, 07743 Jena, Germany. E-mail: andreas.
[email protected] doi: 10.1096/fj.14-268474 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.
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(9). On the one hand, p38 MAPK activates the UPR by regulating the phosphorylation state and/or expression of UPR components such as CHOP and BiP (10, 11). On the other hand, p38 MAPK is activated as consequence of the UPR through inositol-requiring enzyme 1 and PERK oligomerization (12). Because SCD-1 and p38 MAPK are both relevant for cell cycle control and cell stress, we speculated that p38 MAPK might be the missing link between SCD-1 activity and the ER. Our study aimed to investigate the role of p38 MAPK for SCD-1 signal transduction and to elucidate the relevance of this signaling network for cell function, in particular related to the UPR and ER. Indeed, p38 MAPK seemingly mediates the effect of SCD-1 on the ER under specific physiologically relevant stress conditions in fibroblasts. By suppressing excessive p38 MAPK activity, SCD-1 reduced ER stress, attenuated the PERK arm of the UPR, and maintained the structural integrity of the ER upon cell cycle arrest and lipotoxic stress. In contrast to previous studies focusing on the pathophysiological role of SCD-1 substrates (6, 13), our study reveals a major role of SCD-1 products in regulating p38 MAPK activity. Remarkably, the interference with SCD-1 activity is sufficient for activation of p38 MAPK and, if cells are already stressed, to reinforce ER stress and the UPR.
serum (FCS) for 20 hours (37°C, 5% CO2) as described elsewhere (15). Round mitotic cells were detached by rocking and squirting, washed 3 times with ice-cold PBS pH 7.4 and reseeded at 4 3 106 cells/75 cm2 flask. 3T3-L1 fibroblasts were differentiated into adipocytes as described elsewhere (16). Postconfluent cells (12-well plate) were treated with DMEM medium containing 10% FCS, 1 mg/ml insulin, 1 mM dexamethasone, and 0.5 mM 3-isobutyl-1methylxanthine for 2 days (37°C, 5% CO2) and then cultivated for another 2 days in DMEM medium plus 10% FCS and 1 mg/ml insulin. Determination of cell number and cell viability Total and viable cells were counted after trypan blue staining using a Vi-CELL Series Cell Counter (Beckman Coulter, Krefeld, Germany). Alternatively, cell viability was measured using the colorimetric thiazolyl blue tetrazolium bromide (MTT) dye reduction assay. NIH-3T3 cells (7 3 103/96-well plate) were cultivated for 16 h (37°C, 5% CO2) and then preincubated with vehicle (DMSO) or CAY10566 for 48 hours. MTT was added, the formazan product solubilized with SDS (10%, m/v, in 20 mM HCl) after 3 hours, and the absorbance read at 595 nm as described elsewhere (17). Inhibition of SCD-1 by RNA interference
MATERIALS AND METHODS Materials RT-PCR primers were obtained from TIB MOLBIOL (Berlin, Germany). Secondary antibodies were from LI-COR Biosciences (Lincoln, NE, USA). Skepinone-L was synthesized as previously described (14). Materials used: CAY10566, Cayman (Ann Arbor, MI, USA); FlexiTube GeneSolution siRNAs directed against SCD-1 (QIAGEN , Hilden, Germany); nonimmune goat serum, Invitrogen (Carlsbad, CA, USA); DMEM/high glucose (4.5 g/L) medium, trypsin/EDTA solution (PAA Laboratories, Coelbe, Germany); mouse anticyclin D1 (1:2000), rabbit anti-cyclin B1 (1:1000), mouse anticyclin E (1:1000), rabbit or mouse anti-b-actin (1:1000), mouse anti-phospho-ERK1/2 (Thr202/Tyr204; 1:2000), rabbit anti-phospho-MEK1/2 (Ser217/221; 1:1000); rabbit antiphospho-myristoylated alanine-rich C-kinase substrates (Ser152/ 156; 1:1000), rabbit anti-caspase 3 (1:1000), rabbit anti-phosphoSrc family (Tyr416; 1:1000); mouse anti-phospho-JNK (Thr183/ Tyr185; 1:2000), rabbit anti-phospho-Akt (Ser473; 1:1000), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182; 1:1000), rabbit antip38 MAPK (1:1000), mouse anti-IkBa (1:1000), mouse antiCHOP (1:1000), rabbit anti-ATF-4 (1:1000), rabbit anti-BiP (1:1000: Cell Signaling, Danvers, MA, USA); mouse antiendoplasmic reticulum/Golgi intermediate compartment (ERGIC) 53, Enzo Life Sciences (L¨orrach, Germany); lipid standards (Avanti Polar Lipids, Alabaster, AL, USA); 1,2-3H-2deoxy-D-glucose (Hartmann Analytics, Braunschweig, Germany); solvents and all other chemicals were obtained from SigmaAldrich (St. Louis, MO, USA) or Wako Pure Chemicals (Osaka, Japan) unless stated otherwise. Cells, cell differentiation, and cell cycle synchronization Mouse NIH-3T3 and 3T3-L1 fibroblasts were grown at 37°C and 5% CO2 in DMEM medium supplemented with 10% (v/v) heatinactivated FCS. To synchronize NIH-3T3 cells in G2/M-phase of the cell cycle, cells (70–80% confluent) were treated with nocodazole (0.4 mg/ml) in DMEM supplemented with 10% fetal calf
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NIH-3T3 cells were grown to approximately 60% confluence in 25 cm2 dishes and transfected with siRNA duplex oligonucleotides (15 nM) using Lipofectamine RNAiMAX transfection reagent (10 ml, Invitrogen) according to the manufacturer’s protocol. The 3 SCD-1 siRNAs targeted the sequences 1) 59CACAACAGCTTTAAATAATAA-39, 2) 59-TAGTGAGATTTGAATAATTAA-39, and 3) 59-CCGGTACAGTATTCTTATAAA-39, respectively. ON-TARGETplus Non-targeting siRNA #1 (Thermo Scientific, Waltham, MA, USA) was used as scrambled control siRNA. Extraction of lipids Lipids were extracted from NIH-3T3 cells (5 3 105 in PBS pH 7.4) by successive addition of methanol, chloroform, and saline (final ratio: 14:34:35:17) as previously described (18). The organic layer was evaporated, and the extracted lipids were dissolved in 100 ml methanol and diluted, and 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine and 1,2dimyristoyl-sn-glycero-3-phosphatidylethanolamine were used as internal standards. Reversed phase liquid chromatography Lipids were separated on an Acquity UPLC BEH C8 column (1.7 mm, 1 3 100 mm, Waters, Milford, MA, USA) using an Acquity Ultraperformance LC system (Waters) as previously described (15). For phospholipid analysis, chromatography was performed at a flow rate of 0.75 ml/min at 45°C using a gradient of 30% mobile phase A (acetonitrile/water, 10/90, 10 mM ammonium acetate)/70% mobile phase B (acetonitrile/water, 95/5, 10 mM ammonium acetate) to 20% mobile phase A/80% mobile phase B within 5 minutes and to 100% mobile phase B within 2 minutes followed by isocratic elution for another 2 minutes. Triacylglycerols and cholesterol ester were separated at a flow rate of 0.75 ml/min at 45°C using a gradient of 100% mobile phase A (acetonitrile/water, 95:5, 10 mM ammonium acetate) to 70% mobile phase A/20% mobile phase B (isopropanol) within 6 minutes and subsequent isocratic elution for 3 minutes.
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KOEBERLE ET AL.
Mass spectrometry The chromatography system was coupled to a QTRAP 5500 Mass Spectrometer (AB Sciex, Darmstadt, Germany) or a Vantage Triple Stage Quadrupole Mass Spectrometer (Thermo Scientific; for cell cycle analysis). Both were equipped with electrospray ionization sources. Quantification of glycerophospholipids using the QTRAP 5500 Mass Spectrometer was based on the detection of both fatty acid anion fragments through multiple reaction monitoring according to (15). The most intensive transition was selected for quantification. The ion spray voltage was set to 4500 V in the negative ion mode; the heated capillary temperature to 350–700°C; the sheath gas pressure to 45–55 psi; the auxiliary gas pressure to 75–80 psi; the declustering potential to 40–50 V; and the collision energy to 46, 38, 20, 62, and 52 V for phosphatidylcholines, ethanolamines, serines, PI, and phosphatidylglycerols, respectively. Sphingomyelins were quantified as [M + H]+ by m/z = 184 precursor ion scans (collision energy: 33 V). Using the Vantage Triple Stage Quadrupole Mass Spectrometer, cell cycle-dependent changes in the lipid composition were monitored by m/z = 184 precursor ion scans in the positive ion mode (collision energy: 35 V; phosphatidylcholines and sphingomyelins) or full scans in the negative ion mode (all other phospholipids) as described elsewhere (19). Phosphatidylethanolamine and -serine headgroups were confirmed by m = 141.0 or m = 87.0 neutral loss scans (collision energy: 25 V, positive and negative ion mode, respectively) and phosphatidylcholine and PI headgroups by m/z = 184 or m/z = 241.0 precursor ion scans (collision energy: 35 V, positive and negative ion mode, respectively). The fatty acid composition was determined at a collision energy of 40 V by product ion scans. The higher signal intensity of sn-1 than sn-2 fatty acid anions was utilized to estimate the isomeric position of the fatty acids (20). Triacylglycerols and cholesterol ester were analyzed as [M + NH4]+ adducts by multiple reaction monitoring using a QTRAP 5500 Mass Spectrometer. Transitions to [M-fatty acid anion]+ fragments were measured for lipid identification. The most intensive (and species-specific) transition was selected for quantification. The isomeric positions of the fatty acids in triacylglycerols were not determined. In variation to the settings described and referenced above, the ion spray voltage was set to 5500 V in the positive ion mode, the heated capillary temperature to 350–400°C, the sheath gas pressure to 55–60 psi, the auxiliary gas pressure to 70 psi, the declustering potential to 55–120 V, and the collision energy to 35 V (triacylglycerols) or 22 V (cholesterol ester). Mass spectra were processed using the Analyst 1.6 (AB Sciex, Darmstadt, Germany) or Xcalibur 2.0 software (Thermo Scientific) as described elsewhere (15, 20). The proportion of lipid species (=relative intensity) is given as percentage of the sum of all species in the respective subclass (=100%). Total phospholipid subclass intensities combine the intensities of all species of the respective subclass and were normalized to the number of cells and the internal standard 1,2-dimyristoyl-sn-glycero-3phosphatidylcholine.
Sample preparation, SDS-PAGE, and Western blot Cells were sonified (2 3 5 s, on ice) in 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 1 mM phenylmethanesulphonyl fluoride, 60 mg/ml soybean trypsin inhibitor, 10 mg/ml leupeptin, 5 mM sodium fluoride, 1 mM sodium vanadate, and 2.5 mM sodium pyrophosphate. After centrifugation (12,000 g, 5 min, 4°C), the supernatant was taken up in 13 SDS/PAGE sample loading buffer [125 mM Tris-HCl pH 6.5, 25% (m/v) sucrose, 5% SDS (m/v), 0.25% (m/v) bromophenol blue, and 5% (v/v) b-mercaptoethanol] and boiled
P38 MAPK IN SCD-1/ER SIGNALING
for 5 minutes at 95°C. Aliquots (10 mg protein) were resolved by 10 or 12% (m/v) SDS-PAGE and transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare, Munich, Germany). Membranes were blocked with 5% (m/v) BSA or skim milk for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C. Immunoreactive bands were stained with IRDye 800CW-labeled (1:10,000, each) and/or IRDye 680LTlabeled anti-rabbit or anti-mouse IgG (1:80,000, each) and visualized by an Odyssey infrared imager (LI-COR Biosciences). Data from densitometric analysis were background-corrected.
Fatty acid supplementation The culture medium of NIH-3T3 cells was replaced by DMEM medium containing 10% (v/v) heat-inactivated FCS plus palmitate, palmitoleate, or oleate at the indicated concentrations. Culture medium supplemented with 400 mM palmitate was sonicated at 40°C for 20 minutes prior use. Cells were incubated at 37°C and 5% CO2 for the indicated times.
Cellular uptake of 2-deoxy-D-glucose Cellular uptake of 2-deoxy-D-glucose was determined according to Yand and Yao (16). Differentiated 3T3-L1 adipocytes (25 cm2 flask) were treated with CAY10566 (3 mM), Skepinone-L (1 mM), or vehicle (DMSO) for 48 hours (37°C, 5% CO2). Cells were washed, supplemented with vehicle (DMSO), Skepinone-L (1 mM), or the control inhibitor cytochalasin B, and stimulated with insulin (100 nM) in 10 mM HEPES/10 mM sodium phosphate buffer (pH 7.4) containing 128 mM sodium chloride, 4.7 mM potassium chloride, 1.25 mM calcium chloride, and 1.25 mM magnesium sulfate for 15 minutes. Then, 3H-labeled 2-deoxy-D-glucose (0.5 mCi/ml at 0.5 mM) was added. After 10 minutes, cells were washed 3 times with ice-cold PBS pH 7.4 plus 10 mM glucose, immediately lysed in 0.1 N NaOH and mixed with Rotiszint eco plus (3 ml, Carl Roth GmbH, Karlsruhe, Germany) for liquid scintillation counting using a Packard TRI-CARB 2100TR Liquid Scintillation Analyzer.
Immunofluorescence staining and microscopy NIH-3T3 cells (2 3 104/cm2) were seeded onto coverslips and cultured in presence of CAY10566 (3 mM), Skepinone-L (1 mM), or vehicle (DMSO; 37°C, 5% CO2). The culture medium was replaced after 42 hours against culture medium containing palmitate (400 mM) plus CAY10566 (3 mM), Skepinone-L (1 mM), or vehicle (DMSO). This step was omitted for experiments using nonstressed cells. After 6 hours, samples were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 5% normal goat serum (10 minutes, room temperature). Samples were incubated with mouse antiERGIC53 antibody (1:100; Enzo Life Sciences) for 1 hour at room temperature followed by goat anti-mouse IgG Alexa Fluor 555 (1:1000; 1 hour, room temperature; Invitrogen). DNA was stained with 0.1 mg/ml DAPI for 3 minutes at room temperature. The coverslips were mounted on glass slides with Mowiol containing 2.5% n-propyl gallate (Sigma-Aldrich). The fluorescence was visualized with an Axio Observer.Z1 microscope and a Plan-Apochromat 403/1.3 Oil DIC M27 objective (both by Carl Zeiss GmbH, Jena Germany). Images were taken at room temperature with an AxioCam MR3 camera and were acquired, cut, linearly adjusted in the overall brightness and contrast, and exported to TIF by the AxioVision 4.8 software (Carl Zeiss GmbH).
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TABLE 1. Primer sequences used in quantitative RT-PCR experiments Sense primer (59 → 39)
Anti-sense primer (59 → 39)
CATCATTCTCATGGTCCTGCTG GGAGGAACATCATTCTCATGGC GCTGTGCTATGTTGCTCTAGACTT TGACAATGAATACGGCTACAGCA
AGCCGTGCCTTGTAAGTTCTGT AGCCGTGCCTTGTATGTTCTGT AATTGAATGTAGTTTCATGGATGC CTCCTGTTATTATGGGGGTCTGG
Gene
mSCD-1 mSCD-2 mb-actin mGAPDH
experiments or to the amount of RNA for cell cycle studies. Results are given in arbitrary units.
Quantitative RT-PCR Total RNA was prepared using E.Z.N.A. Total RNA Kit I (Omega Bio-tek, Norcross, GA, USA). First-strand cDNAs were synthesized from 1 mg RNA using Superscript III (Invitrogen). PCR was conducted in Mx3000P 96-well plates (25 ml) using a Mx3005P qPCR system (Agilent Technologies, Santa Clara, CA, USA). The PCR mix contained cDNA (2.5 ml), Maxima SYBR Green/Rox qPCR Master Mix (13, Thermo Scientific) and forward and reverse primers (0.5 mM, each). Primer information is provided in Table 1. The cycling program started with 10 minutes at 95°C and included 40 cycles of 15 seconds at 95°C, 30 seconds at 63°C, and 30 seconds at 72°C. cDNA levels were quantified using the MxPro QPCR Software. mRNA expression was normalized to glyceraldehyde-3-phosphate dehydrogenase for knockdown
A
FA-containing PI / total PI [%]
MUFA-PL / total PL [% control]
** ***
75
*** 50 25 0
Data are presented as mean 6 SE of n observations. Statistical evaluation of the data was performed by 1-way ANOVAs for independent or correlated samples followed by Tukey honest significant difference (HSD) post hoc tests or by Student’s t test for paired and correlated samples. P values ,0.05 were considered statistically significant. All statistical calculations were performed using GraphPad InStat 3.10 (GraphPad Software Inc., La Jolla, CA, USA).
C
B
+ CAY 100
Statistics
p-p38 MAPK
*** *** ***
80
p38 MAPK 60
MUFAs saturated FAs
40
*** ***
20
***
0
0.1
1
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D
0
PC PE PS PI PG SM
1 10
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CAY [μM] w/o
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100
***
*** ***
40 20
50 0
0
w/o 1 3 10 CAY [μM]
ctrl #1 #2 #3 siRNA
***
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CAY [μM]
***
100 50 0
ctrlsiRNA
40
p-p38 MAPK
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β-actin
0
ct rl N A C AY
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*
80
R
200
*
PI(18:1/18:1) [% control]
*
SCD-1 mRNA [% control]
p-p38 MAPK density [% control]
80
3
H 100
density [%]
G
F
si
E
1
ctrl #1 #2 #3 siRNA
Figure 1. Inhibition of SCD-1 decreases the proportion of MUFA-containing phospholipids and activates p38 MAPK. A–C) NIH-3T3 cells (5 3 105 cells/25 cm2 flask) were treated with CAY10566 (CAY; 10 mM if not indicated otherwise) or vehicle (DMSO) for 48 h. A) Change of MUFA-containing phospholipids (MUFA-PLs) by CAY; 100% corresponds to relative intensities of 55.5 6 0.5%, 56.1 6 0.4%, 60.5 6 0.4%, 34.4 6 0.5, 98.2 6 0.2%, and 4.9 6 0.2% for MUFA-containing phosphatidylcholine (PC), - ethanolamine (PE), -serine (PS), PI, phosphatidylglycerol (PG), and sphingomyelin (SM), respectively. B) Signal intensities of MUFA- or saturated fatty acid (FA)-containing PI species relative to total PI intensity. C–E, H) Phosphorylation of p38 MAPK (Thr180/Tyr182) was analyzed by Western blot. Controls: p38 MAPK (C) and b-actin (D). Western blots are representative of 3 independent experiments. E, H) Densitometric analysis. F–H) SCD-1 was knocked down in NIH-3T3 cells (60% confluent; 25 cm2 flask) using three different siRNAs (#1–3) for 48 hours. F) SCD-1 mRNA expression normalized to glyceraldehyde-3-phosphate dehydrogenase. G) Cellular proportion of PI(18:1/18:1) after knockdown with siRNAs #1–3. Nontargeting siRNA was transfected as control (ctrl). Data are given as means 6 SE; n = 3–5. *P , 0.05, **P , 0.01, ***P , 0.001 vs. the vehicle control (A–E) or nontargeting siRNA (F–H); ANOVA + Tukey HSD post hoc tests or Student’s t test.
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RESULTS
A
B
CAY
Lowering SCD-1 activity specifically activates p38 MAPK
Supplementation of MUFAs prevents p38 MAPK activation upon SCD-1 inhibition Inhibition of SCD-1 might principally activate p38 MAPK by depleting MUFAs (that are produced by SCD-1) or by accumulating saturated fatty acids (that are SCD-1 substrates). Saturated fatty acids at very high concentrations (400 mM) are well-known activators of p38 MAPK (25), but they failed to enhance p38 MAPK phosphorylation at physiological relevant tissue concentrations P38 MAPK IN SCD-1/ER SIGNALING
β-actin -
16:0 16:1 18:1
C
arsenite
-
16:0 16:1 18:1 fatty acid
-
D
DTT p-p38 MAPK β-actin
-
-
16:0 16:1 18:1
-
-
16:0 16:1 18:1
fatty acid
E p-p38 MAPK β-actin
w /o C ar AY se ni te D D T 16 A2 :0 31 87
To unravel the missing link between SCD-1 activity and ER function, we first investigated the effect of the specific SCD1 inhibitor CAY10566 (21) on major signaling pathways related to proliferation, apoptosis, and cell stress in NIH3T3 mouse fibroblasts. Focus was placed on the stressactivated protein kinases p38 MAPK and JNK, which link cell stress responses to survival and metabolism. SCD activity is inhibited by CAY10566 under our experimental conditions, as shown by the reduced ratio of MUFAs in cellular phospholipids (Fig. 1A). The subgroup of PIs responded most sensitive to CAY10566 (Fig. 1A, B). The decrease of monounsaturated PI was accompanied by an elevated ratio of saturated fatty acids (Fig. 1B, Supplemental Fig. 1A, B), particularly by an increased proportion of the major PI species 1-stearoyl-2-arachidonoyl-snglycero-3-phosphatidylinositol [PI(18:0/20:4)] (Supplemental Fig. 1A). The concomitant increase of arachidonic acid in this particular PI species depends on a redistribution (Supplemental Fig. 1A) but not an enrichment of total arachidonic acid within PI (Supplemental Fig. 1C). The total signal intensity of the PI subgroup remained unchanged (Supplemental Fig. 1D). Inhibition of SCD-1 resulted in a concentration- (EC50 = 1 mM) and timedependent phosphorylation ($24 hours) and thus activation of p38 MAPK as determined by Western blot (Fig. 1C–E, Supplemental Fig. 2A). None of the other signaling pathways investigated were affected by CAY10566 (Supplemental Fig. 2B)—neither the stress-activated JNK cascade nor Akt and ERK signaling, which have previously been suggested to be regulated by SCD-1 (22–24). Among the relevant proteins analyzed were JNK; p38 MAPK; Akt; ERK; cyclins B1, D1, and E; PKC; Src; NF-kB (i.e., IkB); and caspase 3. The negative regulation of p38 MAPK by SCD-1 was confirmed by knockdown of SCD-1. NIH-3T3 cells were transiently transfected with siRNAs either directed against SCD-1 or encoding a scrambled nonmatching sequence as control. All 3 SCD-1 siRNAs inhibited SCD-1 mRNA expression by approximately 40–60% (Fig. 1F) and significantly reduced the proportion of 1,2-dioleoyl-snglycero-3-phosphatidylinositol [PI(18:1/18:1)] (Fig. 1G), which represents a sensitive marker for SCD activity (Supplemental Fig. 1A). As expected, knockdown of SCD-1 significantly enhanced the phosphorylation of p38 MAPK (Fig. 1H).
p-p38 MAPK
Figure 2. MUFAs prevent the activation of p38 MAPK upon inhibition of SCD-1. A, B) NIH-3T3 cells (5 3 105 cells/25 cm2 flask) were treated with vehicle (DMSO) and the free fatty acids palmitate (16:0), palmitoleate (16:1), or oleate (18:1; 10 mM, each) for 48 hours in the absence (A) or presence of CAY10566 (CAY, 3 mM; for 48 hours, B), sodium arsenite (50 mM; for 6 hours, C), and DTT (10 mM) for 6 hours (D). E) Effect of cellular stressors on p38 MAPK phosphorylation. NIH-3T3 cells (5 3 105 cells/25 cm2 flask) were treated with CAY10566 (CAY, 3 mM) for 48 hours or with sodium arsenite (50 mM), DTT (10 mM), palmitate (400 mM), or A23187 (5 mM) for 6 hours. Phosphorylation of p38 MAPK (Thr180/ Tyr182) and expression of b-actin was analyzed by Western blot. Western blots are representative of 3 independent experiments.
(i.e., 10 mM, Fig. 2A), rather excluding a role of saturated fatty acids in activating p38 MAPK by SCD-1 inhibition. However, when SCD-1 was inhibited (and thus the conversion of saturated fatty acids to MUFAs was blocked), low concentrations of saturated fatty acids were sufficient to further augment the activation of p38 MAPK (Fig. 2B). Supplementation of the MUFAs oleate or palmitoleate (10 mM, each) prevented p38 MAPK activation by CAY10566 (Fig. 2B), indicating that the lack of MUFAs rather than an accumulation of saturated fatty acids mediates the effect of SCD-1 on p38 MAPK. Differences between oleate and palmitoleate were not observed (Fig. 2A,B). Supplementation of MUFAs failed to suppress p38 MAPK activation per se. They neither suppressed the basal (Fig. 2A) nor the excessive p38 MAPK phosphorylation induced by genotoxic or metabolic stressors like arsenite (Fig. 2C) or DTT (Fig. 2D). Fig. 2E compares the magnitude of p38 MAPK phosphorylation for the cellular stressors used in this study. 5
The effect of SCD-1 and p38 MAPK on saturated fatty acid-induced ER stress has been investigated in NIH-3T3 cells. Palmitate at pathophysiological concentrations (400 mM) induced the expression of the UPR/ER stress markers BiP, ATF4, and CHOP as well as phosphorylation of p38 MAPK (Fig. 4A-E). These effects of palmitate were not or hardly diminished by Skepinone-L. Concomitant inhibition of SCD-1 and treatment with palmitate further activated the UPR and p38 MAPK. Blockade of p38 MAPK by Skepinone-L almost completely reversed the additional induction of the UPR as consequence of blocking SCD-1 under these stress conditions. Hence, SCD-1 counteracts ER stress and the induction of the UPR by preventing p38 MAPK activation, whereas the induction of the UPR by the SCD-1 substrate and saturated fatty acid palmitate does not critically depend on p38 MAPK (which is in agreement with previous studies) (12). Moreover, the failure of Skepinone-L to suppress the palmitate-dependent activation of the UPR further supports that the lack of SCD-1 products and not the increase in saturated fatty acids transduce the SCD-1 signal to p38 MAPK. ER stress was also induced by using stressors with a principally different mode of action than palmitate (i.e., A23187: disruption of Ca2+ gradients; DTT: denaturation of proteins), but neither did
SCD-1 counteracts palmitate-induced ER stress by reducing p38 MAPK activation After confirming the regulation of p38 MAPK by SCD-1, we addressed the relevance of this cascade for cell function. SCD-1 and p38 MAPK have been reported to regulate neutral lipid biosynthesis (8, 26), cell proliferation (1, 9), glucose uptake (27, 28) and saturated fatty acid-induced ER stress (1, 9). Whether these processes are linearly modulated by SCD-1 through a p38 MAPK-dependent pathway was investigated by rescue experiments using the specific p38 MAPK inhibitor Skepinone-L (14). In accordance with previous studies (8, 26), inhibition of SCD-1 by CAY10566 reduced the cellular content of triacylglycerols (Fig. 3A), impaired cell proliferation and viability in NIH3T3 cells (Fig. 3B–D), and suppressed insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes (Fig. 3E). However, Skepinone-L failed to reverse the CAY10566dependent effects (Fig. 3A, D, E), excluding an involvement of p38 MAPK in mediating the SCD-1 responses on neutral lipid biosynthesis, proliferation, survival, and glucose uptake. Skepinone-L inhibits p38 MAPK with an IC50 of 25–50 nM in diverse cell lines/types including NIH-3T3 mouse fibroblasts as previously reported (14, 15).
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Figure 3. Role of SCD-1 and p38 MAPK for neutral lipid biosynthesis, cell proliferation, and viability and insulin-dependent glucose uptake. NIH-3T3 cells (2 3 104 cells/cm2, A–D) or differentiated 3T3-L1 adipocytes (confluent, E) were treated with vehicle (DMSO), CAY10566 (CAY; 3 mM or as indicated), and/or Skepinone-L (SK-L; 1 mM) for 48 hours. A) Effect on total lipid subclass intensities normalized to the internal standard 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine and the number of cells; 100% is based on relative units. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol; SM, sphingomyelin; TG, triacylglycerol; CE, cholesterol ester. B) Cell numbers were determined using a Vi-CELL Series Cell Counter. 100% corresponds to 5.3 6 0.4 3 104 cells/cm2. C, D) Cell proliferation respectively viability was assessed by the MTT colorimetric assay. E) Cellular uptake of 3H-labeled 2-deoxy-D-glucose (2-DOG; 0.5 mCi/ml). 3T3-L1 preadipocytes or adipocytes were stimulated with insulin (100 nM) in presence of vehicle (DMSO), Skepinone-L (1 mM), or cytochalasin B (25 mM) for 15 min; 100% corresponds to 533 6 49 dpm/min. Data are given as means 6 SE; n = 2–4. *P , 0.05, **P , 0.01, ***P , 0.001 vs. the vehicle (A–D) or insulin-stimulated control (E); ANOVA + Tukey HSD post hoc tests.
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Figure 4. Inhibition of SCD-1 activates the UPR through p38 MAPK in palmitate-stressed cells. NIH-3T3 cells (2 3 104 cells/cm2) were preincubated with vehicle (DMSO), CAY10566 (CAY; 3 mM), and/or Skepinone-L (SK-L; 1mM) for 42 hours and then treated with palmitate (16:0; 400 mM) for 6 hours. A–E) Phosphorylation of p38 MAPK (Thr180/Tyr182) and expression of b-actin, BiP, ATF4, and CHOP was analyzed by Western blot. Western blots are representative of 3–4 independent experiments. B-E) Densitometric analysis. Data are given as means 6 SE; n = 3–4. *P , 0.05, **P , 0.01 vs. the palmitate-treated control; ANOVA + Tukey HSD post hoc tests. F) The ERGIC marker protein ERGIC53 was detected by mouse anti-ERGIC53 and visualized using anti-mouse IgG Alexa Fluor 555 (scale bar, 20 mm). The stains of ERGIC53 are representative for 3 independent experiments and merged with DNA stains (merge; ERGIC53: red, DAPI: blue).
inhibition of SCD-1 enhance the stress response nor did additional inhibition of p38 MAPK show any effect (Supplemental Fig. 3). SCD-1 maintains the integrity of the ERGIC during palmitate-induced ER stress through p38 MAPK ER stress and the UPR are associated with morphological changes of the ER, particularly an expansion to increase its biosynthetic and secretory capacity (25). Whether SCD-1 modulates the ER structure through a p38 MAPKdependent pathway was analyzed by immunofluorescence microscopy after staining for the ERGIC marker protein ERGIC53. The ERGIC of untreated cells showed a diffuse to granular distribution and partially concentrated around the nucleus (Fig. 4F). Addition of palmitate (400 mM) increased the size of the ER/Golgi compartment and evoked a diffuse redistribution of ERGIC53 from the P38 MAPK IN SCD-1/ER SIGNALING
perinuclear region toward the cytoplasm. The perinuclear localization of ERGIC53 was further diminished in the presence of CAY10566 and restored by Skepinone-L almost to the level of untreated cells, indicating a role of p38 MAPK in regulating ER/Golgi integrity by SCD-1. ER morphology was not affected by CAY10566 in nonstressed cells (Fig. 5A), and combined inhibition of SCD-1 and p38 MAPK induced ER expansion (Fig. 5A) along with UPR activation (Fig. 5B, C). Role of SCD-1 and p38 MAPK during the cell cycle and G2/M-phase arrest Induction of cell stress by pathophysiologically relevant concentrations of palmitate or by A23187, DTT, or arsenite induces strong stress responses and is therefore well suited to investigate the signal transduction of SCD-1 and p38 7
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MAPK. It is unclear, however, whether these signaling networks under such harsh conditions are relevant under more physiological situations. Because the cell cycle depends on membrane/ER biogenesis and sensitively responds to cell stress, we studied the physiological relevance of the SCD-1/p38 MAPK/ER-axis during cell cycle arrest and progression. NIH-3T3 cells were synchronized in G2/M-phase with the anti-microtubule agent nocodazole for 20 hours followed by a mitotic shake-off before cells were released into a new cell cycle. Cell cycle progression was monitored by staining the DNA (which duplicates within the cell cycle) with propidium iodide and flow cytometric analysis (15). SCD-1 mRNA expression peaked during early G1-phase as shown by RT-PCR (Fig. 6A). The expression of b-actin, GAPDH, and SCD-2 mRNA was monitored as control and did not significantly change during the cell cycle (Supplemental Fig. 4A–C). The induction of SCD-1 correlated with an accumulation of monounsaturated PI (Fig. 6B). Other phospholipid subgroups than PI were hardly affected (Fig. 6C). p38 MAPK phosphorylation was induced immediately after cell cycle reentry as previously reported (29) and rapidly declined (Fig. 6D, F) when SCD-1 expression increased (Fig. 6A), in accordance with our finding that SCD-1 suppresses p38 MAPK activation. Induction of ER stress (characterized by BiP expression) followed p38 MAPK phosphorylation but remained at a low level (Fig. 6D, G). We speculated that suppression of p38 MAPK by SCD-1 might help to maintain ER homeostasis after G2/M cell cycle arrest. In fact, inhibition of SCD-1 by CAY10566 strongly increased the magnitude and duration of p38 MAPK activation 8
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Figure 5. Role of SCD-1 and p38 MAPK for UPR signaling, ER stress, and ERGIC subcellular distribution in absence of stress inducers. NIH3T3 cells (2 3 104 cells/cm2) were treated with vehicle (DMSO), CAY10566 (CAY, 3 mM), and/ or Skepinone-L (SK-L; 1mM) for 48 hours. A) Coincubation with CAY and SK-L induced the expression of the ERGIC marker protein ERGIC53 (which was stained as described for Fig. 4F). The stains of ERGIC53 are representative for 3 independent experiments and merged with DNA stains (merge; ERGIC53: red, DAPI: blue). Scale bar, 20 mm. B,C) Phosphorylation of p38 MAPK (Thr180/Tyr182) and expression of b-actin, BiP, ATF4, and CHOP was analyzed by Western blot. Western blots are representative of 3 independent experiments. C) Densitometric analysis of CHOP expression. Data are given as means 6 SE; n = 3. **P , 0.01; ANOVA + Tukey HSD post hoc tests.
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(Fig. 6E, F) and induced sustained ER stress during G1- and S-phase (Fig. 6E, G). DISCUSSION The importance of SCD, p38 MAPK, and the UPR for ER homeostasis, cancer, and metabolic diseases has been addressed and confirmed by numerous studies (1, 4, 9), but how these key players interact with each other is poorly understood. We found that a decrease in SCD-1 activity induces the UPR as well as structural changes of the ER through the stress-activated protein kinase p38 MAPK (Fig. 7). We demonstrate that this novel signaling cascade is important to prevent ER stress and to maintain the structure of the ER under lipotoxic stress and upon cell cycle arrest. Changes in the phospholipid composition (as induced by SCD-1) have long been considered to have broad, unspecific effects on multiple membrane proteins. The specificity of signal transmission from SCD-1 to ER homeostasis, which we here describe, would thus be better explained by the existence of lipokine-like SCD-1 metabolites rather than by altered membrane properties. Accordingly, our lipidomic approach provides comprehensive insights into the regulation of the cellular lipid composition by SCD-1 and the cell cycle, thereby suggesting a role of distinct phospholipid-based SCD-1 metabolites as regulators of p38 MAPK signaling. In light of the pleiotropic functions of SCD-1, p38-MAPK, and ER homeostasis, such a mechanism might have broad relevance for diverse cellular processes and diseases.
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It has been speculated that inhibition of SCD-1 induces the UPR by increasing membrane saturation. In fact, drastic changes in the phospholipid saturation index (20–80 mol %), as expected from supplementing high concentrations of palmitate (400 mM), interrupt Ca2+ homoeostasis (5) and activate PERK (6). Along these lines, we found that palmitate at pathophysiological concentrations induces ER stress and the UPR independently from p38 MAPK. More questionable is whether the regulation of SCD-1 is sufficient to achieve the required increase in phospholipid saturation. Deletion or inhibition of SCD-1 shifted the proportion of saturated fatty acids in major phospholipid classes, such as phosphatidylcholines and phosphatidylethanolamines, only by 3–12 mol % as shown in this study (Supplemental Fig. 1B) and by others (2, 7, 8). Alternative mechanisms apparently exist, such as signal transmission via p38 MAPK as here described for fibroblasts under certain stress conditions. In contrast to its effects on phospholipid saturation, changes in SCD-1 activity are sufficient to modulate ER stress through regulating p38 MAPK, in particular by the formation of SCD-1 products. Of interest, we and others recently proposed a lipokine character for the minor MUFA palmitoleate (20, 30). Also the specificity by which SCD-1 inhibition activates p38 MAPK and the UPR might be better explained by lipokine-like SCD-1 products (or metabolites) rather than by general membrane effects due to phospholipid saturation. Thus, 1) SCD-1 exclusively interfered with p38 MAPK signaling among multiple signal transduction pathways P38 MAPK IN SCD-1/ER SIGNALING
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Figure 6. SCD-1 counteracts p38 MAPK activation and ER stress upon G2/M cell cycle arrest. NIH-3T3 cells (4 3 106 cells/75 cm2 flask) were preincubated with vehicle (DMSO) or CAY10566 (CAY; 3 mM) for 48 hours, synchronized by nocodazole (0.4 mg/ml; 0 hours), and released into a new cell cycle. A, B) SCD-1 mRNA expression (A) and the proportion of PIs containing MUFAs (B) peaked during early G1-phase. C) Change of MUFA-containing phospholipids (PLs) in early G1-phase relative to S-phase (3 h vs. 14 h after removal of nocodazole); 100% corresponds to relative intensities of 52.6 6 0.7%, 32.4 6 1.4%, 55.3 6 1.6%, 10.1 6 0.4%, 84.2 6 0.3%, and 7.4 6 0.4% for MUFA-containing phosphatidylcholine (PC), -ethanolamine (PE), -serine (PS), PI, phosphatidylglycerol (PG), and sphingomyelin (SM), respectively. D–G) Phosphorylation of p38 MAPK (Thr180/Tyr182) and BiP was analyzed by Western blot. Expression of b-actin was determined as control. Western blots are representative of 3 independent experiments. F, G) Densitometric analysis. Data are given as means 6 SE; n = 3. *P , 0.05, **P , 0.01, ***P , 0.001 vs. G2/M-phase (0 hours; A, B), S-phase (14 hours; C), or vehicle-treated cells (F, G); ANOVA + Tukey HSD post hoc tests.
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investigated; 2) the negative regulation of p38 MAPK by SCD-1 only affected ER homeostasis, but not other cellular functions; and 3) the regulation of ER homeostasis was limited to distinct stress conditions. Our comprehensive analysis of the phospholipid profile upon interference with SCD-1 and cell cycle progression accordingly revealed a group of SCD-1-derived phospholipids whose proportion is tightly regulated. Especially PIs—a minor phospholipid class with highest abundance in the ER and nucleus of rodent fibroblasts (31)—respond sensitively to alterations in SCD-1-derived MUFAs. The proportion of MUFAcontaining PIs is reduced relative to species with saturated fatty acids, thereby resulting in a redistribution of PUFAs between PI species. These findings are in line with our recent study describing that SCD-1-derived palmitoleate is specifically incorporated into PI in mouse fibroblasts through de novo phospholipid biosynthesis (20). Indirect effects of SCD-1 inhibition on lipid metabolic enzymes (e.g., elongases) have previously been suggested (32) and might contribute to shaping the phospholipid profile. Accordingly, high concentrations of palmitic acid (as used to induce ER stress) activate cytosolic phospholipase A2 and thus deplete arachidonic acid from membrane phospholipids in neuroblastoma cells (33). The accumulation of saturated fatty acids by inhibition of SCD-1 seems, however, not sufficient for a functionally relevant activation of cPLA2 in fibroblasts because the arachidonic acid content of phospholipids is not decreased. Phospholipids have long been neglected as potential lipid mediators and 9
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Figure 7. Link between SCD-1, p38 MAPK, and ER homeostasis in NIH-3T3 cells. SCD-1 converts saturated fatty acids into MUFAs, which represent the major fatty acid species of membrane phospholipids (PLs). SCD-1-derived MUFAs (or their metabolites) counteract p38 MAPK activation under defined stress conditions, particularly during palmitate-induced ER stress and upon G2/M cell cycle arrest. Inhibition of SCD-1 activates p38 MAPK, promotes ER stress, activates the UPR, and induces a redistribution of the ERGIC.
were only recently considered as specific interaction partners of proteins (15, 34). Whether free MUFAs, SCD-1 metabolites, or overall changes in the phospholipid composition mediate the biological effects of SCD-1 is unknown so far. Our hypothesis of a lipokine-like SCD-1 phospholipid metabolite is supported by several lipidomic studies, for which the proportion of monounsaturated phospholipids correlates with cell cycle progression (this study), proliferation (20), and diseases like cancer, diabetes, and obesity (35–37). Moreover, the content of monounsaturated phospholipids is associated with SCD-1 overexpression in human breast cancer (35). How SCD-1derived phospholipid metabolites might interact with p38 MAPK or upstream signaling components is currently investigated in our lab. The regulation of p38 MAPK and ER homeostasis is a key event during diverse physiological processes (9, 25). Our focus was set on the cell cycle, which responds sensitively to changes in lipid biosynthesis and cell stress and requires an intact ER/Golgi secretory network for both protein and lipid biosynthesis (4, 25, 38). Activation of p38 MAPK and induction of the UPR prepare the cell for division but can also arrest the cell cycle at the G1/S- or G2/M-checkpoint (9, 25, 38). These apparently conflicting observations might depend on the magnitude and duration of p38 MAPK and UPR activation, as supported by our data. On the one hand, SCD-1 was strongly induced during early G1phase, suppressed the excessive p38 MAPK activation after a G2/M arrest, and prevented prolonged ER stress during G1/S-phase. These findings resemble what we have observed for palmitate-stressed cells. In both models, SCD-1 protected from ER stress by suppressing unleashed p38 MAPK activation. On the other hand, we found for nonstressed cells that a moderate increase of basal p38 MAPK activity (upon inhibition of SCD-1) protects against ER 10
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stress, which would otherwise be induced due to the lack of SCD-1. Such a regulatory loop might become relevant during the S-phase of the cell cycle, when both SCD-1 and p38 MAPK activities are low. In conclusion, our study 1) demonstrates that SCD-1 negatively regulates the activation of p38 MAPK; 2) confirms the link between SCD-1 and p38 MAPK as relevant regulatory system of the UPR, ER stress, and the ER/Golgi structure under distinct stress conditions; 3) reveals an unexpected specificity of the signal transduction from SCD-1 via p38 MAPK to the ER; 4) presents a group of phospholipid SCD-1 metabolites, which are highly responsive toward changes in SCD-1 activity; 5) challenges the widely distributed assumption that the decrease of saturated fatty acids by SCD-1 underlies its stress-protective functions; and 6) discusses a role of SCD-1-derived phospholipid metabolites as regulators of p38 MAPK activation. It is tempting to speculate that the link between SCD-1, p38 MAPK, and ER homeostasis has a wide range of implications, for example in cancer, diabetes, and obesity, for which major roles of SCD-1, p38 MAPK, and ER stress are established (1, 4, 9). The authors thank Kevin Sch¨omburg and Christoph Seidel for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (KO 4589/4-1 to A.K.); Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to T.S.); Grant-in-Aid for Young Scientists (B) from the MEXT of Japan (to H.S.); the Cell Science Research Foundation (to H.S.); and Takeda Science Foundation (to A.K.).
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