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Coordinated Regulation of the Orosomucoid-like Gene Family Expression Controls de Novo Ceramide Synthesis in Mammalian Cells*

  • Kerstin Kiefer
    Footnotes
    Affiliations
    From the Laboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain and
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  • Amado Carreras-Sureda
    Footnotes
    Affiliations
    From the Laboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain and
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  • Roberto García-López
    Affiliations
    From the Laboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain and
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  • Fanny Rubio-Moscardó
    Affiliations
    From the Laboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain and
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  • Josefina Casas
    Affiliations
    Research Unit on Bioactive Molecules (RUBAM), Department of Biomedicinal Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC), 08034 Barcelona, Spain
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  • Gemma Fabriàs
    Affiliations
    Research Unit on Bioactive Molecules (RUBAM), Department of Biomedicinal Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC), 08034 Barcelona, Spain
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  • Rubén Vicente
    Correspondence
    To whom correspondence should be addressed: Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain. Tel.: 34-933160854; Fax: 34-933160901; E-mail: [email protected]
    Affiliations
    From the Laboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain and
    Search for articles by this author
  • Author Footnotes
    * This work was supported by Spanish Ministry of Economy and Competitiveness Grants SAF2010-16725 and SAF2011-22444, Fondo de Investigación Sanitaria Red HERACLES Grant RD12/0042/0014, Fondos Europeos de Desarrollo Regional, Generalitat de Catalunya Grant SGR2014, and Fundació la Marató de TV3 Grant 20134030.
    1 Both authors contributed equally to this work.
Open AccessPublished:December 17, 2014DOI:https://doi.org/10.1074/jbc.M114.595116
      The orosomucoid-like (ORMDL) protein family is involved in the regulation of de novo sphingolipid synthesis, calcium homeostasis, and unfolded protein response. Single nucleotide polymorphisms (SNPs) that increase ORMDL3 expression have been associated with various immune/inflammatory diseases, although the pathophysiological mechanisms underlying this association are poorly understood. ORMDL proteins are claimed to be inhibitors of the serine palmitoyltransferase (SPT). However, it is not clear whether individual ORMDL expression levels have an impact on ceramide synthesis. The present study addressed the interaction with and regulation of SPT activity by ORMDLs to clarify their pathophysiological relevance. We have measured ceramide production in HEK293 cells incubated with palmitate as a direct substrate for SPT reaction. Our results showed that a coordinated overexpression of the three isoforms inhibits the enzyme completely, whereas individual ORMDLs are not as effective. Immunoprecipitation and fluorescence resonance energy transfer (FRET) studies showed that mammalian ORMDLs form oligomeric complexes that change conformation depending on cellular sphingolipid levels. Finally, using macrophages as a model, we demonstrate that mammalian cells modify ORMDL genes expression levels coordinately to regulate the de novo ceramide synthesis pathway. In conclusion, we have shown a physiological modulation of SPT activity by general ORMDL expression level regulation. Moreover, because single ORMDL3 protein alteration produces an incomplete inhibition of SPT activity, this work argues against the idea that ORMDL3 pathophysiology could be explained by a simple on/off mechanism on SPT activity.
      Background: Knockdown of orosomucoid-like (ORMDL) proteins releases serine palmitoyltransferase (SPT) activity.
      Results: Significant changes in SPT activity were detected when all three ORMDLs were overexpressed. Sphingolipids do not modify SPT-ORMDL interaction but rearrange ORMDLs. Macrophages suppress ORMDLs to induce de novo ceramide synthesis.
      Conclusion: Coordinated ORMDL expression regulation strongly influences SPT activity.
      Significance: SPT-ORMDL complex presents transcriptional and post-translational regulation.

      Introduction

      Orosomucoid-like (ORMDL)
      The abbreviations used are: ORMDL
      orosomucoid-like
      SPT
      serine palmitoyltransferase
      CFP
      cyan fluorescent protein
      PA
      palmitate.
      proteins belong to a family of transmembrane proteins that contains three members located in the endoplasmic reticulum (
      • Hjelmqvist L.
      • Tuson M.
      • Marfany G.
      • Herrero E.
      • Balcells S.
      • Gonzàlez-Duarte R.
      ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins.
      ). The association of ORMDL3 with childhood asthma in a genome-wide association study (
      • Moffatt M.F.
      • Kabesch M.
      • Liang L.
      • Dixon A.L.
      • Strachan D.
      • Heath S.
      • Depner M.
      • von Berg A.
      • Bufe A.
      • Rietschel E.
      • Heinzmann A.
      • Simma B.
      • Frischer T.
      • Willis-Owen S.A.
      • Wong K.C.
      • Illig T.
      • Vogelberg C.
      • Weiland S.K.
      • von Mutius E.
      • Abecasis G.R.
      • Farrall M.
      • Gut I.G.
      • Lathrop G.M.
      • Cookson W.O.
      Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma.
      ) stimulated renewed interest in the study of these proteins. Despite the absence of the single nucleotide polymorphism (SNP) identified in the genome-wide association study (rs 7216389) in a coding region of the gene, this first study correlated increased expression of ORMDL3 with the risk allele. Since then, several SNPs around the ORMDL3 gene that are associated with pathologies like inflammatory bowel disease, type I diabetes, and rheumatoid arthritis have been described (
      • McGovern D.P.
      • Gardet A.
      • Törkvist L.
      • Goyette P.
      • Essers J.
      • Taylor K.D.
      • Neale B.M.
      • Ong R.T.
      • Lagacé C.
      • Li C.
      • Green T.
      • Stevens C.R.
      • Beauchamp C.
      • Fleshner P.R.
      • Carlson M.
      • D'Amato M.
      • Halfvarson J.
      • Hibberd M.L.
      • Lördal M.
      • Padyukov L.
      • Andriulli A.
      • Colombo E.
      • Latiano A.
      • Palmieri O.
      • Bernard E.J.
      • Deslandres C.
      • Hommes D.W.
      • de Jong D.J.
      • Stokkers P.C.
      • Weersma R.K.
      • NIDDK IBD Genetics Consortium
      • Sharma Y.
      • Silverberg M.S.
      • Cho J.H.
      • Wu J.
      • Roeder K.
      • Brant S.R.
      • Schumm L.P.
      • Duerr R.H.
      • Dubinsky M.C.
      • Glazer N.L.
      • Haritunians T.
      • Ippoliti A.
      • Melmed G.Y.
      • Siscovick D.S.
      • Vasiliauskas E.A.
      • Targan S.R.
      • Annese V.
      • Wijmenga C.
      • Pettersson S.
      • Rotter J.I.
      • Xavier R.J.
      • Daly M.J.
      • Rioux J.D.
      • Seielstad M.
      Genome-wide association identifies multiple ulcerative colitis susceptibility loci.
      ,
      • Barrett J.C.
      • Hansoul S.
      • Nicolae D.L.
      • Cho J.H.
      • Duerr R.H.
      • Rioux J.D.
      • Brant S.R.
      • Silverberg M.S.
      • Taylor K.D.
      • Barmada M.M.
      • Bitton A.
      • Dassopoulos T.
      • Datta L.W.
      • Green T.
      • Griffiths A.M.
      • Kistner E.O.
      • Murtha M.T.
      • Regueiro M.D.
      • Rotter J.I.
      • Schumm L.P.
      • Steinhart A.H.
      • Targan S.R.
      • Xavier R.J.
      • NIDDK IBD Genetics Consortium
      • Libioulle C.
      • Sandor C.
      • Lathrop M.
      • Belaiche J.
      • Dewit O.
      • Gut I.
      • Heath S.
      • Laukens D.
      • Mni M.
      • Rutgeerts P.
      • Van Gossum A.
      • Zelenika D.
      • Franchimont D.
      • Hugot J.P.
      • de Vos M.
      • Vermeire S.
      • Louis E.
      • Belgian-French IBD Consortium, Wellcome Trust Case Control Consortium
      • Cardon L.R.
      • Anderson C.A.
      • Drummond H.
      • Nimmo E.
      • Ahmad T.
      • Prescott N.J.
      • Onnie C.M.
      • Fisher S.A.
      • Marchini J.
      • Ghori J.
      • Bumpstead S.
      • Gwilliam R.
      • Tremelling M.
      • Deloukas P.
      • Mansfield J.
      • Jewell D.
      • Satsangi J.
      • Mathew C.G.
      • Parkes M.
      • Georges M.
      • Daly M.J.
      Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease.
      ,
      • Barrett J.C.
      • Clayton D.G.
      • Concannon P.
      • Akolkar B.
      • Cooper J.D.
      • Erlich H.A.
      • Julier C.
      • Morahan G.
      • Nerup J.
      • Nierras C.
      • Plagnol V.
      • Pociot F.
      • Schuilenburg H.
      • Smyth D.J.
      • Stevens H.
      • Todd J.A.
      • Walker N.M.
      • Rich S.S.
      Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes.
      ,
      • Kurreeman F.A.
      • Stahl E.A.
      • Okada Y.
      • Liao K.
      • Diogo D.
      • Raychaudhuri S.
      • Freudenberg J.
      • Kochi Y.
      • Patsopoulos N.A.
      • Gupta N.
      • CLEAR investigators
      • Sandor C.
      • Bang S.Y.
      • Lee H.S.
      • Padyukov L.
      • Suzuki A.
      • Siminovitch K.
      • Worthington J.
      • Gregersen P.K.
      • Hughes L.B.
      • Reynolds R.J.
      • Bridges Jr., S.L.
      • Bae S.C.
      • Yamamoto K.
      • Plenge R.M.
      Use of a multiethnic approach to identify rheumatoid-arthritis-susceptibility loci, 1p36 and 17q12.
      ).
      The genome-wide association study approach to the diagnosis of genetic risk factors is not focused on precandidate genes, making it an excellent tool to identify new genes involved in diseases. However, sometimes the identified genes have ill defined functions as in the case of ORMDL3 at the time it was detected. To elucidate the pathophysiology associated with ORMDL3, several laboratories have been trying to understand the role of ORMDLs in cell physiology. Our laboratory has focused on the effects of ORMDL3 expression levels in calcium homeostasis, a likely connection between an endoplasmic reticulum-resident protein and immune system dysfunction. We have found that the expression levels of this protein are inversely correlated with the calcium content of the endoplasmic reticulum due to an inhibition of sarco/endoplasmic reticulum Ca2+-ATPase pump activity (
      • Cantero-Recasens G.
      • Fandos C.
      • Rubio-Moscardo F.
      • Valverde M.A.
      • Vicente R.
      The asthma-associated ORMDL3 gene product regulates endoplasmic reticulum-mediated calcium signaling and cellular stress.
      ). In addition, we have shown that the store-operated calcium entry, the main calcium entry pathway during T cell activation, is altered because ORMDL3 decreases the calcium buffering capacity of the mitochondria and the subsequent calcium-dependent inactivation of the calcium release-activated Ca2+ channel (
      • Carreras-Sureda A.
      • Cantero-Recasens G.
      • Rubio-Moscardo F.
      • Kiefer K.
      • Peinelt C.
      • Niemeyer B.A.
      • Valverde M.A.
      • Vicente R.
      ORMDL3 modulates store-operated calcium entry and lymphocyte activation.
      ).
      Conversely, it has been shown that the presence of ORMDLs acts as a break for the de novo sphingolipid synthesis pathway (
      • Han S.
      • Lone M.A.
      • Schneiter R.
      • Chang A.
      Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control.
      ,
      • Breslow D.K.
      • Collins S.R.
      • Bodenmiller B.
      • Aebersold R.
      • Simons K.
      • Shevchenko A.
      • Ejsing C.S.
      • Weissman J.S.
      Orm family proteins mediate sphingolipid homeostasis.
      ). In yeast and mammalian cells, the complete knockdown of ORMDLs releases serine palmitoyltransferase (SPT) activity and generates an increase in long chain bases and ceramides. The expression of any of the isoforms in this knockdown condition rescues the normal functioning of the pathway (
      • Breslow D.K.
      • Collins S.R.
      • Bodenmiller B.
      • Aebersold R.
      • Simons K.
      • Shevchenko A.
      • Ejsing C.S.
      • Weissman J.S.
      Orm family proteins mediate sphingolipid homeostasis.
      ). This fact, together with the interaction between the yeast ORMDL isoforms (Orms) and the SPT enzyme, has established the idea that ORMDLs are the endogenous inhibitors of SPT. In addition, the SPT-Orm interaction is dependent on a phosphorylation reaction that disrupts an oligomeric complex of Orms and interferes with SPT-Orm interaction (
      • Breslow D.K.
      • Collins S.R.
      • Bodenmiller B.
      • Aebersold R.
      • Simons K.
      • Shevchenko A.
      • Ejsing C.S.
      • Weissman J.S.
      Orm family proteins mediate sphingolipid homeostasis.
      ). The regulation of the pathway implicated in Orm phosphorylation and its sensitivity to ceramide cell content have been described in yeast (
      • Roelants F.M.
      • Breslow D.K.
      • Muir A.
      • Weissman J.S.
      • Thorner J.
      Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae.
      ). However, the role of this phosphorylation in SPT-ORMDL interaction is not clear in mammals because the N-terminal regulatory region described in yeasts is absent in mammalian ORMDLs. More remarkable is the lack of evidence that different ORMDL3 expression levels in mammalian cells alter SPT activity; this is a critical gap in understanding the pathophysiology associated with this gene.
      We herein evaluate the role of mammalian ORMDLs in the ceramide synthesis context with three specific aims: (i) to explore the effect of ORMDL3 overexpression on SPT activity, (ii) to study the ORMDL-SPT complex interaction and its dependence on ceramide cell content, and (iii) to find a physiological context in which cells modify ORMDL expression levels to modulate SPT activity. For this purpose, we used HEK293 cells as the heterologous expression system and palmitate treatment to stimulate SPT activity. We performed coimmunoprecipitation studies between SPT-ORMDL complex elements and FRET studies to confirm and explore conformational changes. Moreover, we used the RAW264.7 monocytic cell line to study the regulation of ORMDL expression during de novo sphingolipid generation under the activation process.

      DISCUSSION

      Complete absence of ORMDL protein expression in mammalian and yeast cells leads to an overproduction of ceramides through the de novo synthesis pathway (
      • Han S.
      • Lone M.A.
      • Schneiter R.
      • Chang A.
      Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control.
      ,
      • Breslow D.K.
      • Collins S.R.
      • Bodenmiller B.
      • Aebersold R.
      • Simons K.
      • Shevchenko A.
      • Ejsing C.S.
      • Weissman J.S.
      Orm family proteins mediate sphingolipid homeostasis.
      ,
      • Siow D.L.
      • Wattenberg B.W.
      Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis.
      ). This fact, together with the positive coimmunoprecipitation of ORMDLs and SPT, supports the scenario in which ORMDLs are endogenous inhibitors of the first enzyme of de novo sphingolipid synthesis. Conversely, the rescue of normal growth on the Orm1/Orm2 double KO yeast strain by the expression of mammalian ORMDL3 isoform has generated the idea that all the ORMDL members have redundant functions and that this is a regulatory mechanism conserved throughout evolution (
      • Breslow D.K.
      • Collins S.R.
      • Bodenmiller B.
      • Aebersold R.
      • Simons K.
      • Shevchenko A.
      • Ejsing C.S.
      • Weissman J.S.
      Orm family proteins mediate sphingolipid homeostasis.
      ). In addition to the different regulatory elements described in yeast, it has been shown that ORMDLs participate in the cascade that senses and inhibits SPT depending on the intracellular ceramide content (
      • Roelants F.M.
      • Breslow D.K.
      • Muir A.
      • Weissman J.S.
      • Thorner J.
      Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae.
      ,
      • Siow D.L.
      • Wattenberg B.W.
      Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis.
      ). However, the current knowledge regarding ORMDL function in controlling sphingolipid synthesis still has many blanks that hamper the understanding of the pathophysiological role of ORMDL3, a gene implicated as a risk factor in several diseases (
      • Moffatt M.F.
      • Kabesch M.
      • Liang L.
      • Dixon A.L.
      • Strachan D.
      • Heath S.
      • Depner M.
      • von Berg A.
      • Bufe A.
      • Rietschel E.
      • Heinzmann A.
      • Simma B.
      • Frischer T.
      • Willis-Owen S.A.
      • Wong K.C.
      • Illig T.
      • Vogelberg C.
      • Weiland S.K.
      • von Mutius E.
      • Abecasis G.R.
      • Farrall M.
      • Gut I.G.
      • Lathrop G.M.
      • Cookson W.O.
      Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma.
      ,
      • McGovern D.P.
      • Gardet A.
      • Törkvist L.
      • Goyette P.
      • Essers J.
      • Taylor K.D.
      • Neale B.M.
      • Ong R.T.
      • Lagacé C.
      • Li C.
      • Green T.
      • Stevens C.R.
      • Beauchamp C.
      • Fleshner P.R.
      • Carlson M.
      • D'Amato M.
      • Halfvarson J.
      • Hibberd M.L.
      • Lördal M.
      • Padyukov L.
      • Andriulli A.
      • Colombo E.
      • Latiano A.
      • Palmieri O.
      • Bernard E.J.
      • Deslandres C.
      • Hommes D.W.
      • de Jong D.J.
      • Stokkers P.C.
      • Weersma R.K.
      • NIDDK IBD Genetics Consortium
      • Sharma Y.
      • Silverberg M.S.
      • Cho J.H.
      • Wu J.
      • Roeder K.
      • Brant S.R.
      • Schumm L.P.
      • Duerr R.H.
      • Dubinsky M.C.
      • Glazer N.L.
      • Haritunians T.
      • Ippoliti A.
      • Melmed G.Y.
      • Siscovick D.S.
      • Vasiliauskas E.A.
      • Targan S.R.
      • Annese V.
      • Wijmenga C.
      • Pettersson S.
      • Rotter J.I.
      • Xavier R.J.
      • Daly M.J.
      • Rioux J.D.
      • Seielstad M.
      Genome-wide association identifies multiple ulcerative colitis susceptibility loci.
      ,
      • Barrett J.C.
      • Hansoul S.
      • Nicolae D.L.
      • Cho J.H.
      • Duerr R.H.
      • Rioux J.D.
      • Brant S.R.
      • Silverberg M.S.
      • Taylor K.D.
      • Barmada M.M.
      • Bitton A.
      • Dassopoulos T.
      • Datta L.W.
      • Green T.
      • Griffiths A.M.
      • Kistner E.O.
      • Murtha M.T.
      • Regueiro M.D.
      • Rotter J.I.
      • Schumm L.P.
      • Steinhart A.H.
      • Targan S.R.
      • Xavier R.J.
      • NIDDK IBD Genetics Consortium
      • Libioulle C.
      • Sandor C.
      • Lathrop M.
      • Belaiche J.
      • Dewit O.
      • Gut I.
      • Heath S.
      • Laukens D.
      • Mni M.
      • Rutgeerts P.
      • Van Gossum A.
      • Zelenika D.
      • Franchimont D.
      • Hugot J.P.
      • de Vos M.
      • Vermeire S.
      • Louis E.
      • Belgian-French IBD Consortium, Wellcome Trust Case Control Consortium
      • Cardon L.R.
      • Anderson C.A.
      • Drummond H.
      • Nimmo E.
      • Ahmad T.
      • Prescott N.J.
      • Onnie C.M.
      • Fisher S.A.
      • Marchini J.
      • Ghori J.
      • Bumpstead S.
      • Gwilliam R.
      • Tremelling M.
      • Deloukas P.
      • Mansfield J.
      • Jewell D.
      • Satsangi J.
      • Mathew C.G.
      • Parkes M.
      • Georges M.
      • Daly M.J.
      Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease.
      ,
      • Barrett J.C.
      • Clayton D.G.
      • Concannon P.
      • Akolkar B.
      • Cooper J.D.
      • Erlich H.A.
      • Julier C.
      • Morahan G.
      • Nerup J.
      • Nierras C.
      • Plagnol V.
      • Pociot F.
      • Schuilenburg H.
      • Smyth D.J.
      • Stevens H.
      • Todd J.A.
      • Walker N.M.
      • Rich S.S.
      Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes.
      ,
      • Kurreeman F.A.
      • Stahl E.A.
      • Okada Y.
      • Liao K.
      • Diogo D.
      • Raychaudhuri S.
      • Freudenberg J.
      • Kochi Y.
      • Patsopoulos N.A.
      • Gupta N.
      • CLEAR investigators
      • Sandor C.
      • Bang S.Y.
      • Lee H.S.
      • Padyukov L.
      • Suzuki A.
      • Siminovitch K.
      • Worthington J.
      • Gregersen P.K.
      • Hughes L.B.
      • Reynolds R.J.
      • Bridges Jr., S.L.
      • Bae S.C.
      • Yamamoto K.
      • Plenge R.M.
      Use of a multiethnic approach to identify rheumatoid-arthritis-susceptibility loci, 1p36 and 17q12.
      ). The present study tries to cast light on the relationship between mammalian ORMDLs and SPT.
      Until now it was not known whether single variations in ORMDL3 expression could cause changes in SPT function and cellular sphingolipid content. This information is important to understand whether SNPs that exert a cis regulation on ORMDL3 gene expression, claimed to be risk factors of several pathologies (
      • Moffatt M.F.
      • Kabesch M.
      • Liang L.
      • Dixon A.L.
      • Strachan D.
      • Heath S.
      • Depner M.
      • von Berg A.
      • Bufe A.
      • Rietschel E.
      • Heinzmann A.
      • Simma B.
      • Frischer T.
      • Willis-Owen S.A.
      • Wong K.C.
      • Illig T.
      • Vogelberg C.
      • Weiland S.K.
      • von Mutius E.
      • Abecasis G.R.
      • Farrall M.
      • Gut I.G.
      • Lathrop G.M.
      • Cookson W.O.
      Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma.
      ,
      • Verlaan D.J.
      • Berlivet S.
      • Hunninghake G.M.
      • Madore A.M.
      • Larivière M.
      • Moussette S.
      • Grundberg E.
      • Kwan T.
      • Ouimet M.
      • Ge B.
      • Hoberman R.
      • Swiatek M.
      • Dias J.
      • Lam K.C.
      • Koka V.
      • Harmsen E.
      • Soto-Quiros M.
      • Avila L.
      • Celedón J.C.
      • Weiss S.T.
      • Dewar K.
      • Sinnett D.
      • Laprise C.
      • Raby B.A.
      • Pastinen T.
      • Naumova A.K.
      Allele-specific chromatin remodeling in the ZPBP2/GSDMB/ORMDL3 locus associated with the risk of asthma and autoimmune disease.
      ), could be linked to the function of this protein in sphingolipid synthesis. Our results show that increased expression levels of each different ORMDL or the three isoforms together do not affect the ceramide content of the cell. Interestingly, triple overexpression blocks SPT function when stimulating the de novo ceramide synthesis with palmitate. These findings can only be explained by additional regulatory mechanisms besides changes in expression. Thus, ORMDLs would not be allowed to lower the homeostatic content of ceramides in the cell, but their presence suffices to block SPT when ceramides increase to putatively deleterious levels. This effect is observed only in triple overexpression, meaning that all three members are needed for an effective sensing and blockage of the de novo pathway. Nevertheless, our results on the effect of ORMDL expression on SPT activity have to be cautiously interpreted as they may be partially dependent on the experimental settings, i.e. cell type and palmitate treatment. In this sense, we have modified exclusively ORMDL expression levels to mimic, in our in vitro model, as much as possible the pathophysiological context related to the diseases in which ORMDL3 has been implicated. Genetic analysis of ORMDL3-related inflammatory diseases only pointed to changes in ORMDL3 expression without information about possible changes in other elements participating in the SPT-ORMDL complex. In this context and based on the fact that overexpression of ORMDL3 alone did not modify ceramide content and palmitate-induced sphingolipid synthesis, our results do not support a pathophysiological mechanism based on the complete SPT inhibition. Besides, deletion of the first 16 amino acids of the ORMDL3 N-terminal tail has been shown to abolish its impact on calcium signaling (
      • Carreras-Sureda A.
      • Cantero-Recasens G.
      • Rubio-Moscardo F.
      • Kiefer K.
      • Peinelt C.
      • Niemeyer B.A.
      • Valverde M.A.
      • Vicente R.
      ORMDL3 modulates store-operated calcium entry and lymphocyte activation.
      ). However, the lack of differences regarding ceramide content and production observed in the present work for overexpression of this mutant and wild type ORMDL3 argues against an explanation for the effect of ORMDL3 on calcium homeostasis based on its role in sphingolipid synthesis.
      Another aspect we have addressed in this work is the dynamic regulation of ORMDL-SPTLC complexes that has been proposed in light of the experiments performed in yeasts (
      • Breslow D.K.
      • Collins S.R.
      • Bodenmiller B.
      • Aebersold R.
      • Simons K.
      • Shevchenko A.
      • Ejsing C.S.
      • Weissman J.S.
      Orm family proteins mediate sphingolipid homeostasis.
      ). Our results demonstrate that the regulation based on interaction of the yeast ORMDL isoforms is lost in mammalian ORMDLs. The immunoprecipitation experiments did not support the occurrence of changes in the ORMDL-ORMDL or ORMDL-SPTLC interaction when the ceramide content of the cell was manipulated. Interestingly, another regulatory mechanism based on ORMDL phosphorylation via the TORC1 pathway that does not modify the interaction of ORMDL oligomers necessary for complex sphingolipid formation has been found in yeast (
      • Shimobayashi M.
      • Oppliger W.
      • Moes S.
      • Jenö P.
      • Hall M.N.
      TORC1-regulated protein kinase Npr1 phosphorylates Orm to stimulate complex sphingolipid synthesis.
      ). Whether this modulation is similar to the conformational changes observed in our experiments is a question that requires further investigation. It is also important to note that the regulation described in yeast is based on the phosphorylation of the N-terminal part of yeast Orms (
      • Breslow D.K.
      • Collins S.R.
      • Bodenmiller B.
      • Aebersold R.
      • Simons K.
      • Shevchenko A.
      • Ejsing C.S.
      • Weissman J.S.
      Orm family proteins mediate sphingolipid homeostasis.
      ,
      • Roelants F.M.
      • Breslow D.K.
      • Muir A.
      • Weissman J.S.
      • Thorner J.
      Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae.
      ). This domain is only present in yeast and plants, whereas vertebrate proteins cluster differently (
      • Hjelmqvist L.
      • Tuson M.
      • Marfany G.
      • Herrero E.
      • Balcells S.
      • Gonzàlez-Duarte R.
      ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins.
      ). Regarding the stoichiometry of the complex, we propose that ORMDLs are able to form homo- and hetero-oligomers with similar affinity because in our experiments we always overexpressed all the ORMDL members to avoid artificial interactions. Hetero-oligomerization likely results in a slightly different conformation based on the different FRET observed. Finally, our results also suggest that ORMDL regulation of SPT might be achieved by structural rearrangements depending on the intracellular ceramide content that do not involve changes in ORMDL-SPTLC binding affinity.
      Finally, we demonstrate for the first time using physiological stimuli that mammalian cells modulate ceramide content by modifying expression levels of the ORMDL family. We have focused on macrophages where de novo ceramide synthesis has been characterized during LPS activation (
      • Schilling J.D.
      • Machkovech H.M.
      • He L.
      • Sidhu R.
      • Fujiwara H.
      • Weber K.
      • Ory D.S.
      • Schaffer J.E.
      Palmitate and lipopolysaccharide trigger synergistic ceramide production in primary macrophages.
      ,
      • Andreyev A.Y.
      • Fahy E.
      • Guan Z.
      • Kelly S.
      • Li X.
      • McDonald J.G.
      • Milne S.
      • Myers D.
      • Park H.
      • Ryan A.
      • Thompson B.M.
      • Wang E.
      • Zhao Y.
      • Brown H.A.
      • Merrill A.H.
      • Raetz C.R.
      • Russell D.W.
      • Subramaniam S.
      • Dennis E.A.
      Subcellular organelle lipidomics in TLR-4-activated macrophages.
      ,
      • Chang Z.Q.
      • Lee S.Y.
      • Kim H.J.
      • Kim J.R.
      • Kim S.J.
      • Hong I.K.
      • Oh B.C.
      • Choi C.S.
      • Goldberg I.J.
      • Park T.S.
      Endotoxin activates de novo sphingolipid biosynthesis via nuclear factor κB-mediated upregulation of Sptlc2.
      ). The increase in ceramides through the SPT pathway is necessary for correct activation, and its blockage affects several processes such as autophagy and interleukin production (
      • Sims K.
      • Haynes C.A.
      • Kelly S.
      • Allegood J.C.
      • Wang E.
      • Momin A.
      • Leipelt M.
      • Reichart D.
      • Glass C.K.
      • Sullards M.C.
      • Merrill Jr., A.H.
      Kdo2-lipid A, a TLR4-specific agonist, induces de novo sphingolipid biosynthesis in RAW264.7 macrophages, which is essential for induction of autophagy.
      ,
      • Yu H.
      • Valerio M.
      • Bielawski J.
      Fenretinide inhibited de novo ceramide synthesis and proinflammatory cytokines induced by Aggregatibacter actinomycetemcomitans.
      ). In this context, we show that expression of the three ORMDL members is repressed at early time points in correlation with ceramide synthesis induction. The reduction in the ORMDL/SPTLC ratio suggests a regulatory scenario in which ceramide synthesis is induced by reducing the expression of the inhibitory subunits of the SPT complex. These results agree with the heterologous expression analysis where the triple knockdown of ORMDLs is necessary to release SPT activity (
      • Siow D.L.
      • Wattenberg B.W.
      Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis.
      ).
      Our work reinforces the idea that ORMDLs are negative modulators of SPT activity, providing physiological evidence for this role. However, increased individual expression of these proteins does not reduce the ceramide content in the cell, which conflicts with the idea that the pathophysiological link between disease-associated SNPs that change ORMDL3 expression is related to changes in cellular sphingolipid content due to a complete inhibition of the de novo pathway. This also implies that alternative mechanisms should be considered in determining the role of ORMDLs in immune/inflammatory diseases, i.e. the control of calcium homeostasis (
      • Cantero-Recasens G.
      • Fandos C.
      • Rubio-Moscardo F.
      • Valverde M.A.
      • Vicente R.
      The asthma-associated ORMDL3 gene product regulates endoplasmic reticulum-mediated calcium signaling and cellular stress.
      ,
      • Carreras-Sureda A.
      • Cantero-Recasens G.
      • Rubio-Moscardo F.
      • Kiefer K.
      • Peinelt C.
      • Niemeyer B.A.
      • Valverde M.A.
      • Vicente R.
      ORMDL3 modulates store-operated calcium entry and lymphocyte activation.
      ) and the unfolded protein response (
      • Miller M.
      • Tam A.B.
      • Cho J.Y.
      • Doherty T.A.
      • Pham A.
      • Khorram N.
      • Rosenthal P.
      • Mueller J.L.
      • Hoffman H.M.
      • Suzukawa M.
      • Niwa M.
      • Broide D.H.
      ORMDL3 is an inducible lung epithelial gene regulating metalloproteases, chemokines, OAS, and ATF6.
      ,
      • Miller M.
      • Rosenthal P.
      • Beppu A.
      • Mueller J.L.
      • Hoffman H.M.
      • Tam A.B.
      • Doherty T.A.
      • McGeough M.D.
      • Pena C.A.
      • Suzukawa M.
      • Niwa M.
      • Broide D.H.
      ORMDL3 transgenic mice have increased airway remodeling and airway responsiveness characteristic of asthma.
      ).

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