Advertisement

The prohibitin complex regulates macrophage fatty acid composition, plasma membrane packing, and lipid raft-mediated inflammatory signaling

Published:January 18, 2023DOI:https://doi.org/10.1016/j.plefa.2023.102540

      Highlights

      • Role of prohibitins (PHBs) in macrophage signaling was studied using PHB knockdown.
      • PHB knockdown decreases expression and signaling of lipid raft-dependent receptors.
      • Plasma membrane packing and raft formation are altered in PHB knockdown macrophages.
      • PHB controls the composition of cellular fatty acids.
      • PHBs impact macrophage signaling by regulating lipid rafts.

      Abstract

      Prohibitins (PHB1 and PHB2) are ubiquitously expressed proteins which play critical roles in multiple biological processes, and together form the ring-like PHB complex found in phospholipid-rich cellular compartments including lipid rafts. Recent studies have implicated PHB1 as a mediator of fatty acid transport as well as a membrane scaffold mediating B lymphocyte and mast cell signal transduction. However, the specific role of PHBs in the macrophage have not been characterized, including their role in fatty acid uptake and lipid raft-mediated inflammatory signaling. We hypothesized that the PHB complex regulates macrophage inflammatory signaling through the formation of lipid rafts. To evaluate our hypothesis, RAW 264.7 macrophages were transduced with shRNA against PHB1, PHB2, or scrambled control (Scr), and then stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-α), which activate lipid raft-dependent receptor signaling (CD14/TLR4 and TNFR1, respectively). PHB1 knockdown was lethal, whereas PHB2 knockdown (PHB2kd), which also resulted in decreased PHB1 expression, led to attenuated nuclear factor-kappa-B (NF-κB) activation and subsequent cytokine and chemokine production. PHB2kd macrophages also had decreased cell surface TNFR1, CD14, TLR4, and lipid raft marker ganglioside GM1 at baseline and post-stimuli. Post-LPS, PHB2kd macrophages did not increase the concentration of cellular saturated, monounsaturated, and polyunsaturated fatty acids. This was accompanied by decreased lipid raft formation and modified plasma membrane molecular packing, further supporting the PHB complex's importance in lipid raft formation. Taken together, these data suggest a critical role for PHBs in regulating macrophage inflammatory signaling via maintenance of fatty acid composition and lipid raft structure.

      Summary

      Prohibitins are proteins found in phospholipid-rich cellular compartments, including lipid rafts, that play important roles in signaling, transcription, and multiple other cell functions. Macrophages are key cells in the innate immune response and the presence of membrane lipid rafts is integral to signal transduction, but the role of prohibitins in macrophage lipid rafts and associated signaling is unknown. To address this question, prohibitin knockdown macrophages were generated and responses to lipopolysaccharide and tumor necrosis factor-alpha, which act through lipid raft-dependent receptors, were analyzed. Prohibitin knockdown macrophages had significantly decreased cytokine and chemokine production, transcription factor activation, receptor expression, lipid raft assembly and membrane packing, and altered fatty acid remodeling. These data indicate a novel role for prohibitins in macrophage inflammatory signaling through regulation of fatty acid composition and lipid raft formation.

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Prostaglandins, Leukotrienes and Essential Fatty Acids
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Carroll R.G.
        • Zaslona Z.
        • Galvan-Pena S.
        • et al.
        An unexpected link between fatty acid synthase and cholesterol synthesis in proinflammatory macrophage activation.
        J. Biol. Chem. 2018; 293: 5509-5521https://doi.org/10.1074/jbc.RA118.001921
        • Freitas Filho E.G.
        • Jaca L.A.M.
        • Baeza L.C.
        • et al.
        Proteomic analysis of lipid rafts from RBL-2H3 mast cells.
        Int. J. Mol. Sci. 2019; 20: 3904-3925https://doi.org/10.3390/ijms20163904
        • Levental I.
        • Veatch S.
        The continuing mystery of lipid rafts.
        J. Mol. Biol. 2016; 428: 4749-4764https://doi.org/10.1016/j.jmb.2016.08.022
        • Chowdhury S.M.
        • Zhu X.
        • Aloor J.J.
        • et al.
        Proteomic analysis of ABCA1-null macrophages reveals a role for stomatin-like protein-2 in raft composition and toll-like receptor signaling.
        Mol. Cell. Proteomics. 2015; 14: 1859-1870https://doi.org/10.1074/mcp.M114.045179
        • Fessler M.B.
        • Parks J.S.
        Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling.
        J. Immunol. 2011; 187: 1529-1535https://doi.org/10.4049/jimmunol.1100253
        • Pike L.J.
        • Casey L.
        Cholesterol levels modulate EGF receptor-mediated signaling by altering receptor function and trafficking.
        Biochemistry. 2002; 41: 10315-10322https://doi.org/10.1021/bi025943i
        • Lee A.G.
        How lipids affect the activities of integral membrane proteins.
        Biochim. Biophys. Acta. 2004; 1666: 62-87https://doi.org/10.1016/j.bbamem.2004.05.012
        • Epand R.M.
        Do proteins facilitate the formation of cholesterol-rich domains?.
        Biochim. Biophys. Acta. 2004; 1666: 227-238https://doi.org/10.1016/j.bbamem.2004.07.004
        • Legler D.F.
        • Micheau O.
        • Doucey M.A.
        • Tschopp J.
        • Bron C.
        Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NF-kappaB activation.
        Immunity. 2003; 18: 655-664https://doi.org/10.1016/s1074-7613(03)00092-x
        • Pfeiffer A.
        • Bottcher A.
        • Orso E.
        • et al.
        Lipopolysaccharide and ceramide docking to CD14 provokes ligand-specific receptor clustering in rafts.
        Eur. J. Immunol. 2001; 31: 3153-3164https://doi.org/10.1002/1521-4141(200111)31:11<3153::aid-immu3153>3.0.co;2-0
        • Triantafilou M.
        • Miyake K.
        • Golenbock D.T.
        • Triantafilou K.
        Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.
        J. Cell Sci. 2002; 115: 2603-2611https://doi.org/10.1242/jcs.115.12.2603
        • Triantafilou M.
        • Morath S.
        • Mackie A.
        • Hartung T.
        • Triantafilou K.
        Lateral diffusion of Toll-like receptors reveals that they are transiently confined within lipid rafts on the plasma membrane.
        J. Cell Sci. 2004; 117: 4007-4014https://doi.org/10.1242/jcs.01270
        • Motoyama K.
        • Arima H.
        • Nishimoto Y.
        • Miyake K.
        • Hirayama F.
        • Uekama K.
        Involvement of CD14 in the inhibitory effects of dimethyl-alpha-cyclodextrin on lipopolysaccharide signaling in macrophages.
        FEBS Lett. 2005; 579: 1707-1714https://doi.org/10.1016/j.febslet.2005.01.076
        • Wong S.W.
        • Kwon M.J.
        • Choi A.M.
        • Kim H.P.
        • Nakahira K.
        • Hwang D.H.
        Fatty acids modulate Toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner.
        J. Biol. Chem. 2009; 284: 27384-27392https://doi.org/10.1074/jbc.M109.044065
        • Browman D.T.
        • Hoegg M.B.
        • Robbins S.M.
        The SPFH domain-containing proteins: more than lipid raft markers.
        Trends Cell Biol. 2007; 17: 394-402https://doi.org/10.1016/j.tcb.2007.06.005
        • Merkwirth C.
        • Dargazanli S.
        • Tatsuta T.
        • et al.
        Prohibitins control cell proliferation and apoptosis by regulating OPA1-dependent cristae morphogenesis in mitochondria.
        Genes Dev. 2008; 22: 476-488https://doi.org/10.1101/gad.460708
        • Theiss A.L.
        • Idell R.D.
        • Srinivasan S.
        • et al.
        Prohibitin protects against oxidative stress in intestinal epithelial cells.
        FASEB J. 2007; 21: 197-206https://doi.org/10.1096/fj.06-6801com
        • Ande S.R.
        • Xu Y.X.Z.
        • Mishra S.
        Prohibitin: a potential therapeutic target in tyrosine kinase signaling.
        Signal Transduct. Target Ther. 2017; 2: 17059-17066https://doi.org/10.1038/sigtrans.2017.59
        • Chowdhury D.
        • Kumar D.
        • Bhadra U.
        • Devi T.A.
        • Bhadra M.P.
        Prohibitin confers cytoprotection against ISO-induced hypertrophy in H9c2 cells via attenuation of oxidative stress and modulation of Akt/Gsk-3beta signaling.
        Mol. Cell. Biochem. 2017; 425: 155-168https://doi.org/10.1007/s11010-016-2870-3
        • Merkwirth C.
        • Martinelli P.
        • Korwitz A.
        • et al.
        Loss of prohibitin membrane scaffolds impairs mitochondrial architecture and leads to tau hyperphosphorylation and neurodegeneration.
        PLos Genet. 2012; 8e1003021https://doi.org/10.1371/journal.pgen.1003021
        • Merkwirth C.
        • Langer T.
        Prohibitin function within mitochondria: essential roles for cell proliferation and cristae morphogenesis.
        Biochim. Biophys. Acta. 2009; 1793: 27-32https://doi.org/10.1016/j.bbamcr.2008.05.013
        • Berger K.H.
        • Yaffe M.P.
        Prohibitin family members interact genetically with mitochondrial inheritance components in Saccharomyces cerevisiae.
        Mol. Cell. Biol. 1998; 18: 4043-4052https://doi.org/10.1128/MCB.18.7.4043
        • Artal-Sanz M.
        • Tsang W.Y.
        • Willems E.M.
        • et al.
        The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans.
        J. Biol. Chem. 2003; 278: 32091-32099https://doi.org/10.1074/jbc.M304877200
        • Kasashima K.
        • Ohta E.
        • Kagawa Y.
        • Endo H.
        Mitochondrial functions and estrogen receptor-dependent nuclear translocation of pleiotropic human prohibitin 2.
        J. Biol. Chem. 2006; 281: 36401-36410https://doi.org/10.1074/jbc.M605260200
        • He B.
        • Feng Q.
        • Mukherjee A.
        • et al.
        A repressive role for prohibitin in estrogen signaling.
        Mol. Endocrinol. 2008; 22: 344-360https://doi.org/10.1210/me.2007-0400
      1. The Scripps Research Institute. BioGPS website. (2019) Gene expression chart PHB (prohibitin). http://biogps.org/#goto=genereport&id=5245.

        • Kamal A.H.M.
        • Aloor J.J.
        • Fessler M.B.
        • Chowdhury S.M.
        Cross-linking proteomics indicates effects of simvastatin on the TLR2 interactome and reveals ACTR1A as a novel regulator of the TLR2 signal cascade.
        Mol. Cell. Proteomics. 2019; 18: 1732-1744https://doi.org/10.1074/mcp.RA119.001377
        • Kamal A.H.M.
        • Chakrabarty J.K.
        • Udden S.M.N.
        • Zaki M.H.
        • Chowdhury S.M.
        Inflammatory proteomic network analysis of statin-treated and lipopolysaccharide-activated macrophages.
        Sci. Rep. 2018; 8: 164-176https://doi.org/10.1038/s41598-017-18533-1
        • Lucas C.R.
        • Cordero-Nieves H.M.
        • Erbe R.S.
        • et al.
        Prohibitins and the cytoplasmic domain of CD86 cooperate to mediate CD86 signaling in B lymphocytes.
        J. Immunol. 2013; 190: 723-736https://doi.org/10.4049/jimmunol.1201646
        • Kim D.K.
        • Kim H.S.
        • Kim A.R.
        • et al.
        The scaffold protein prohibitin is required for antigen-stimulated signaling in mast cells.
        Sci. Signal. 2013; 6: ra80https://doi.org/10.1126/scisignal.2004098
        • Yurugi H.
        • Tanida S.
        • Ishida A.
        • et al.
        Expression of prohibitins on the surface of activated T cells.
        Biochem. Biophys. Res. Commun. 2012; 420: 275-280https://doi.org/10.1016/j.bbrc.2012.02.149
        • Terashima M.
        • Kim K.M.
        • Adachi T.
        • et al.
        The IgM antigen receptor of B lymphocytes is associated with prohibitin and a prohibitin-related protein.
        EMBO J. 1994; 13: 3782-3792https://doi.org/10.1002/j.1460-2075.1994.tb06689.x
        • Vessal M.
        • Mishra S.
        • Moulik S.
        • Murphy L.J.
        Prohibitin attenuates insulin-stimulated glucose and fatty acid oxidation in adipose tissue by inhibition of pyruvate carboxylase.
        FEBS J. 2006; 273: 568-576https://doi.org/10.1111/j.1742-4658.2005.05090.x
        • Daquinag A.C.
        • Gao Z.
        • Fussell C.
        • et al.
        Fatty acid mobilization from adipose tissue is mediated by CD36 posttranslational modifications and intracellular trafficking.
        JCI Insight. 2021; 6e147057https://doi.org/10.1172/jci.insight.147057
        • Salameh A.
        • Daquinag A.C.
        • Staquicini D.I.
        • et al.
        Prohibitin/annexin 2 interaction regulates fatty acid transport in adipose tissue.
        JCI Insight. 2016; 1: e86351https://doi.org/10.1172/jci.insight.86351
        • Gao Z.
        • Daquinag A.C.
        • Yu Y.
        • Kolonin M.G.
        Endothelial prohibitin mediates bidirectional long-chain fatty acid transport in white and brown adipose tissues.
        DiabetesDiabetes. 2022; 71: 1400-1409https://doi.org/10.2337/db21-0972
        • Wu D.
        • Jian C.
        • Peng Q.
        • et al.
        Prohibitin 2 deficiency impairs cardiac fatty acid oxidation and causes heart failure.
        Cell Death. Dis. 2020; 11: 181-194https://doi.org/10.1038/s41419-020-2374-7
        • Gao Z.
        • Daquinag A.C.
        • Fussell C.
        • Djehal A.
        • Desaubry L.
        • Kolonin M.G.
        Prohibitin inactivation in adipocytes results in reduced lipid metabolism and adaptive thermogenesis impairment.
        Diabetes. 2021; 70: 2204-2212https://doi.org/10.2337/db21-0094
        • Varshney P.
        • Yadav V.
        • Saini N.
        Lipid rafts in immune signalling: current progress and future perspective.
        Immunology. 2016; 149: 13-24https://doi.org/10.1111/imm.12617
        • Madenspacher J.H.
        • Morrell E.D.
        • Gowdy K.M.
        • et al.
        Cholesterol 25-hydroxylase promotes efferocytosis and resolution of lung inflammation.
        JCI Insight. 2020; 5e137189https://doi.org/10.1172/jci.insight.137189
        • Boulter N.
        • Suarez F.G.
        • Schibeci S.
        • et al.
        A simple, accurate and universal method for quantification of PCR.
        BMC Biotechnol. 2016; 16: 27-40https://doi.org/10.1186/s12896-016-0256-y
        • Shaikh S.R.
        • Rockett B.D.
        • Salameh M.
        • Carraway K.
        Docosahexaenoic acid modifies the clustering and size of lipid rafts and the lateral organization and surface expression of MHC class I of EL4 cells.
        J. Nutr. 2009; 139: 1632-1639https://doi.org/10.3945/jn.109.108720
        • Bronkema S.M.
        • Rowntree J.E.
        • Jain R.
        • Schweihofer J.P.
        • Bitler C.A.
        • Fenton J.I.
        A nutritional survey of commercially available grass-finished beef.
        Meat Muscle Biol. 2019; 3: 116-126https://doi.org/10.22175/mmb2018.10.0034
        • Sergin S.
        • Goeden T.
        • Krusinski L.
        • et al.
        Fatty acid and antioxidant composition of conventional compared to pastured eggs: characterization of conjugated linoleic acid and branched chain fatty acid isomers in eggs.
        ACS Food Sci. Technol. 2021; 1: 260-267https://doi.org/10.1021/acsfoodscitech.0c00093
        • Kramer J.K.G.
        • Hernandez M.
        • Cruz-Hernandez C.
        • Kraft J.
        • Dugan M.E.R.
        Combining results of two GC separations partly achieves determination of all cis and trans 16:1, 18:1, 18:2 and 18:3 except CLA isomers of milk fat as demonstrated using Ag-ion SPE fractionation.
        Lipids. 2008; 43: 259-273https://doi.org/10.1007/s11745-007-3143-4
        • Teague H.
        • Harris M.
        • Fenton J.
        • Lallemand P.
        • Shewchuk B.M.
        • Shaikh S.R.
        Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity.
        J. Lipid Res. 2014; 55: 1420-1433https://doi.org/10.1194/jlr.M049809
        • Blank C.
        • Luz A.
        • Bendigs S.
        • Erdmann A.
        • Wagner H.
        • Heeg K.
        Superantigen and endotoxin synergize in the induction of lethal shock.
        Eur. J. Immunol. 1997; 27: 825-833https://doi.org/10.1002/eji.1830270405
        • Morrison D.C.
        • Ryan J.L.
        Endotoxins and disease mechanisms.
        Annu. Rev. Med. 1987; 38: 417-432https://doi.org/10.1146/annurev.me.38.020187.002221
        • Ko K.S.
        • Tomasi M.L.
        • Iglesias-Ara A.
        • et al.
        Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice.
        Hepatology. 2010; 52: 2096-2108https://doi.org/10.1002/hep.23919
        • Wu C.
        • Liu C.
        • Luo K.
        • Li Y.
        • Jiang J.
        • Yan F.
        Changes in expression of the membrane receptors CD14, MHC-II, SR-A, and TLR4 in tissue-specific monocytes/macrophages following porphyromonas gingivalis-LPS stimulation.
        Inflammation. 2018; 41: 418-431https://doi.org/10.1007/s10753-017-0698-y
        • Liu T.
        • Zhang L.
        • Joo D.
        • Sun S.C.
        NF-kappaB signaling in inflammation.
        Signal Transduct. Target Ther. 2017; 2: e17023https://doi.org/10.1038/sigtrans.2017.23
        • Bjorkbacka H.
        • Fitzgerald K.A.
        • Huet F.
        • et al.
        The induction of macrophage gene expression by LPS predominantly utilizes Myd88-independent signaling cascades.
        Physiol. Genomics. 2004; 19: 319-330https://doi.org/10.1152/physiolgenomics.00128.2004
        • McCarthy G.M.
        • Bridges C.R.
        • Blednov Y.A.
        • Harris R.A.
        CNS cell-type localization and LPS response of TLR signaling pathways.
        F1000Res. 2017; 6: 1144-1162https://doi.org/10.12688/f1000research.12036.1
        • Bradley J.R.
        TNF-mediated inflammatory disease.
        J. Pathol. 2008; 214: 149-160https://doi.org/10.1002/path.2287
        • Fritsch J.
        • Zingler P.
        • Sarchen V.
        • Heck A.L.
        • Schutze S.
        Role of ubiquitination and proteolysis in the regulation of pro- and anti-apoptotic TNF-R1 signaling.
        Biochim. Biophys. Acta Mol. Cell Res. 2017; 1864: 2138-2146https://doi.org/10.1016/j.bbamcr.2017.07.017
        • Zhu X.
        • Owen J.S.
        • Wilson M.D.
        • et al.
        Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
        J. Lipid Res. 2010; 51: 3196-3206https://doi.org/10.1194/jlr.M006486
        • Wu Q.
        • Wu S.
        The role of lipid raft translocation of prohibitin in regulation of Akt and Raf-protected apoptosis of HaCaT cells upon ultraviolet B irradiation.
        Mol. Carcinog. 2017; 56: 1789-1797https://doi.org/10.1002/mc.22636
        • Wang K.
        • Liu C.Y.
        • Zhang X.J.
        • et al.
        miR-361-regulated prohibitin inhibits mitochondrial fission and apoptosis and protects heart from ischemia injury.
        Cell Death Differ. 2015; 22: 1058-1068https://doi.org/10.1038/cdd.2014.200
        • Li J.
        • Aung L.H.
        • Long B.
        • Qin D.
        • An S.
        • Li P.
        miR-23a binds to p53 and enhances its association with miR-128 promoter.
        Sci. Rep. 2015; 5: 16422-16434https://doi.org/10.1038/srep16422
        • Kang T.
        • Lu W.
        • Xu W.
        • et al.
        MicroRNA-27 (miR-27) targets prohibitin and impairs adipocyte differentiation and mitochondrial function in human adipose-derived stem cells.
        J. Biol. Chem. 2013; 288: 34394-34402https://doi.org/10.1074/jbc.M113.514372
        • Lai L.
        • Azzam K.M.
        • Lin W.C.
        • et al.
        MicroRNA-33 regulates the innate immune response via ATP binding cassette transporter-mediated remodeling of membrane microdomains.
        J. Biol. Chem. 2016; 291: 19651-19660https://doi.org/10.1074/jbc.M116.723056
        • Gamble S.C.
        • Chotai D.
        • Odontiadis M.
        • et al.
        Prohibitin, a protein downregulated by androgens, represses androgen receptor activity.
        Oncogene. 2007; 26: 1757-1768https://doi.org/10.1038/sj.onc.1209967
        • He B.
        • Kim T.H.
        • Kommagani R.
        • et al.
        Estrogen-regulated prohibitin is required for mouse uterine development and adult function.
        Endocrinology. 2011; 152: 1047-1056https://doi.org/10.1210/en.2010-0732
        • Meyer zum Bueschenfelde C.O.
        • Unternaehrer J.
        • Mellman I.
        • Bottomly K.
        Regulated recruitment of MHC class II and costimulatory molecules to lipid rafts in dendritic cells.
        J. Immunol. 2004; 173: 6119-6124https://doi.org/10.4049/jimmunol.173.10.6119
        • de Bus I.A.
        • America A.H.P.
        • de Ruijter N.C.A.
        • et al.
        PUFA-Derived N-Acylethanolamide probes identify peroxiredoxins and small GTPases as molecular targets in LPS-stimulated RAW264.7 macrophages.
        ACS Chem. Biol. 2022; 17: 2054-2064https://doi.org/10.1021/acschembio.1c00355
        • Wassal S.R.
        • Leng X.
        • Canner S.W.
        • et al.
        Docosahexaenoic acid regulates the formation of lipid rafts: a unified view from experiment and simulation.
        Biochim. Biophys. Acta Biomembr. 2018; 198501993https://doi.org/10.1016/j.bbamem.2018.04.016
        • Shaikh S.R.
        • Wassall S.R.
        • Brown D.A.
        • Kosaraju R.
        N-3 polyunsaturated lipid microclusters, and vitamin E.
        Curr. Top. Membr. 2015; : 209-231https://doi.org/10.1016/bs.ctm.2015.03.003
        • Moreno-Altamirano M.M.
        • Aguilar-Carmona I.
        • Sanchez-Garcia F.J.
        Expression of GM1, a marker of lipid rafts, defines two subsets of human monocytes with differential endocytic capacity and lipopolysaccharide responsiveness.
        Immunology. 2007; 120: 536-543https://doi.org/10.1111/j.1365-2567.2006.02531.x
        • Lingwood D.
        • Ries J.
        • Schwille P.
        • Simons K.
        Plasma membranes are poised for activation of raft phase coalescence at physiological temperature.
        Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 10005-10010https://doi.org/10.1073/pnas.0804374105