Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages
Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polari...
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| Veröffentlicht in: | International journal of molecular sciences 2023-06, Vol.24 (11), p.9641 |
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| creator | Muller, Ittai B Lin, Marry Jonge, Robert de Will, Nico López-Navarro, Baltasar Laken, Conny van der Struys, Eduard A Oudejans, Cees B M Assaraf, Yehuda G Cloos, Jacqueline Puig-Kröger, Amaya Jansen, Gerrit |
| description | Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy. |
| doi_str_mv | 10.3390/ijms24119641 |
| format | Article |
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Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.</description><identifier>ISSN: 1422-0067</identifier><identifier>ISSN: 1661-6596</identifier><identifier>EISSN: 1422-0067</identifier><identifier>DOI: 10.3390/ijms24119641</identifier><identifier>PMID: 37298590</identifier><language>eng</language><publisher>Switzerland: MDPI AG</publisher><subject>Accumulation ; Alternative Splicing ; Analysis ; Annotations ; Arthritis ; Autoimmune diseases ; Cell cycle ; Chemokines ; DNA methylation ; Energy metabolism ; Enzymatic activity ; Enzyme activity ; Enzymes ; Exposure ; Folic acid ; Folic Acid - metabolism ; Folic Acid - pharmacology ; Gene Expression ; Genes ; Genetic engineering ; Genetic research ; Granulocyte-macrophage colony-stimulating factor ; Granulocyte-Macrophage Colony-Stimulating Factor - genetics ; Granulocyte-Macrophage Colony-Stimulating Factor - metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology ; Histones ; Humans ; Immune system ; Inflammation ; Macrophage colony-stimulating factor ; Macrophage Colony-Stimulating Factor - metabolism ; Macrophages ; Macrophages - metabolism ; Metabolic pathways ; Metabolism ; Methotrexate ; Methotrexate - metabolism ; Methotrexate - pharmacology ; Monocytes ; Monocytes - metabolism ; Peptide Synthases - genetics ; Physiological aspects ; Rheumatoid arthritis ; Rheumatoid factor ; RNA ; RNA Precursors - metabolism ; Sequence analysis ; Vitamin B</subject><ispartof>International journal of molecular sciences, 2023-06, Vol.24 (11), p.9641</ispartof><rights>COPYRIGHT 2023 MDPI AG</rights><rights>2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2023 by the authors. 2023</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c437t-cd959a4f7cedd7d06dc7d7efee075b8ed5e5b11a73bac3eb777a3812764211913</cites><orcidid>0000-0001-9150-8026 ; 0000-0002-4811-5419 ; 0000-0003-3211-2982 ; 0000-0001-8190-8368 ; 0000-0003-2943-9757</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10253671/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10253671/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37298590$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Muller, Ittai B</creatorcontrib><creatorcontrib>Lin, Marry</creatorcontrib><creatorcontrib>Jonge, Robert de</creatorcontrib><creatorcontrib>Will, Nico</creatorcontrib><creatorcontrib>López-Navarro, Baltasar</creatorcontrib><creatorcontrib>Laken, Conny van der</creatorcontrib><creatorcontrib>Struys, Eduard A</creatorcontrib><creatorcontrib>Oudejans, Cees B M</creatorcontrib><creatorcontrib>Assaraf, Yehuda G</creatorcontrib><creatorcontrib>Cloos, Jacqueline</creatorcontrib><creatorcontrib>Puig-Kröger, Amaya</creatorcontrib><creatorcontrib>Jansen, Gerrit</creatorcontrib><title>Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages</title><title>International journal of molecular sciences</title><addtitle>Int J Mol Sci</addtitle><description>Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.</description><subject>Accumulation</subject><subject>Alternative Splicing</subject><subject>Analysis</subject><subject>Annotations</subject><subject>Arthritis</subject><subject>Autoimmune diseases</subject><subject>Cell cycle</subject><subject>Chemokines</subject><subject>DNA methylation</subject><subject>Energy metabolism</subject><subject>Enzymatic activity</subject><subject>Enzyme activity</subject><subject>Enzymes</subject><subject>Exposure</subject><subject>Folic acid</subject><subject>Folic Acid - metabolism</subject><subject>Folic Acid - pharmacology</subject><subject>Gene Expression</subject><subject>Genes</subject><subject>Genetic engineering</subject><subject>Genetic research</subject><subject>Granulocyte-macrophage colony-stimulating factor</subject><subject>Granulocyte-Macrophage Colony-Stimulating Factor - genetics</subject><subject>Granulocyte-Macrophage Colony-Stimulating Factor - metabolism</subject><subject>Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology</subject><subject>Histones</subject><subject>Humans</subject><subject>Immune system</subject><subject>Inflammation</subject><subject>Macrophage colony-stimulating factor</subject><subject>Macrophage Colony-Stimulating Factor - metabolism</subject><subject>Macrophages</subject><subject>Macrophages - metabolism</subject><subject>Metabolic pathways</subject><subject>Metabolism</subject><subject>Methotrexate</subject><subject>Methotrexate - metabolism</subject><subject>Methotrexate - pharmacology</subject><subject>Monocytes</subject><subject>Monocytes - metabolism</subject><subject>Peptide Synthases - genetics</subject><subject>Physiological aspects</subject><subject>Rheumatoid arthritis</subject><subject>Rheumatoid factor</subject><subject>RNA</subject><subject>RNA Precursors - metabolism</subject><subject>Sequence analysis</subject><subject>Vitamin B</subject><issn>1422-0067</issn><issn>1661-6596</issn><issn>1422-0067</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>BENPR</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNptkk1v1DAQhiMEoqVw44wiceFAij-SOD6h1dJdkLqiUoGr5diTXRfHTu3squWv8Gdx2FK2CPng8fiZ15rXk2UvMTqllKN35qqPpMSY1yV-lB3jkpACoZo9PoiPsmcxXiFEKKn40-yIMsKbiqPj7OcKxo0fA9zIEfKL4Hf-O8T8g4mDDFNq4e20JUy23prY50twkJ_dDAFiNN7l0ul8ZkcITo5mB_nlYI0ybp0bl7D8m9n5fOWdV7djUp7o5aqYXy6K3_E-vEivBPMD0lmq4IeNXEN8nj3ppI3w4m4_yb4uzr7MPxbnn5ef5rPzQpWUjYXSvOKy7JgCrZlGtVZMM-gAEKvaBnQFVYuxZLSVikLLGJO0wYTVJUm2YXqSvd_rDtu2B63AjUFaMQTTy3ArvDTi4Y0zG7H2O4ERqWjNJoU3dwrBX28hjqI3UYG10oHfRkEaUtZNw1md0Nf_oFd-m6yzewqVNW_QX2otLQjjuvRFUk2iYsYqwlLjjCfq9D9UWhp6o7yDzqT8g4K3-4LkcYwBuvsmMRLTNInDaUr4q0Nj7uE_40N_AfRuxrM</recordid><startdate>20230601</startdate><enddate>20230601</enddate><creator>Muller, Ittai B</creator><creator>Lin, Marry</creator><creator>Jonge, Robert de</creator><creator>Will, Nico</creator><creator>López-Navarro, Baltasar</creator><creator>Laken, Conny van der</creator><creator>Struys, Eduard A</creator><creator>Oudejans, Cees B M</creator><creator>Assaraf, Yehuda G</creator><creator>Cloos, Jacqueline</creator><creator>Puig-Kröger, Amaya</creator><creator>Jansen, Gerrit</creator><general>MDPI AG</general><general>MDPI</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>MBDVC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0001-9150-8026</orcidid><orcidid>https://orcid.org/0000-0002-4811-5419</orcidid><orcidid>https://orcid.org/0000-0003-3211-2982</orcidid><orcidid>https://orcid.org/0000-0001-8190-8368</orcidid><orcidid>https://orcid.org/0000-0003-2943-9757</orcidid></search><sort><creationdate>20230601</creationdate><title>Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages</title><author>Muller, Ittai B ; Lin, Marry ; Jonge, Robert de ; Will, Nico ; López-Navarro, Baltasar ; Laken, Conny van der ; Struys, Eduard A ; Oudejans, Cees B M ; Assaraf, Yehuda G ; Cloos, Jacqueline ; Puig-Kröger, Amaya ; Jansen, Gerrit</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c437t-cd959a4f7cedd7d06dc7d7efee075b8ed5e5b11a73bac3eb777a3812764211913</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Accumulation</topic><topic>Alternative Splicing</topic><topic>Analysis</topic><topic>Annotations</topic><topic>Arthritis</topic><topic>Autoimmune diseases</topic><topic>Cell cycle</topic><topic>Chemokines</topic><topic>DNA methylation</topic><topic>Energy metabolism</topic><topic>Enzymatic activity</topic><topic>Enzyme activity</topic><topic>Enzymes</topic><topic>Exposure</topic><topic>Folic acid</topic><topic>Folic Acid - 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Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.</abstract><cop>Switzerland</cop><pub>MDPI AG</pub><pmid>37298590</pmid><doi>10.3390/ijms24119641</doi><orcidid>https://orcid.org/0000-0001-9150-8026</orcidid><orcidid>https://orcid.org/0000-0002-4811-5419</orcidid><orcidid>https://orcid.org/0000-0003-3211-2982</orcidid><orcidid>https://orcid.org/0000-0001-8190-8368</orcidid><orcidid>https://orcid.org/0000-0003-2943-9757</orcidid><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1422-0067 |
| ispartof | International journal of molecular sciences, 2023-06, Vol.24 (11), p.9641 |
| issn | 1422-0067 1661-6596 1422-0067 |
| language | eng |
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| source | MDPI - Multidisciplinary Digital Publishing Institute; MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central |
| subjects | Accumulation Alternative Splicing Analysis Annotations Arthritis Autoimmune diseases Cell cycle Chemokines DNA methylation Energy metabolism Enzymatic activity Enzyme activity Enzymes Exposure Folic acid Folic Acid - metabolism Folic Acid - pharmacology Gene Expression Genes Genetic engineering Genetic research Granulocyte-macrophage colony-stimulating factor Granulocyte-Macrophage Colony-Stimulating Factor - genetics Granulocyte-Macrophage Colony-Stimulating Factor - metabolism Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology Histones Humans Immune system Inflammation Macrophage colony-stimulating factor Macrophage Colony-Stimulating Factor - metabolism Macrophages Macrophages - metabolism Metabolic pathways Metabolism Methotrexate Methotrexate - metabolism Methotrexate - pharmacology Monocytes Monocytes - metabolism Peptide Synthases - genetics Physiological aspects Rheumatoid arthritis Rheumatoid factor RNA RNA Precursors - metabolism Sequence analysis Vitamin B |
| title | Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-05T17%3A40%3A11IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Methotrexate%20Provokes%20Disparate%20Folate%20Metabolism%20Gene%20Expression%20and%20Alternative%20Splicing%20in%20Ex%20Vivo%20Monocytes%20and%20GM-CSF-%20and%20M-CSF-Polarized%20Macrophages&rft.jtitle=International%20journal%20of%20molecular%20sciences&rft.au=Muller,%20Ittai%20B&rft.date=2023-06-01&rft.volume=24&rft.issue=11&rft.spage=9641&rft.pages=9641-&rft.issn=1422-0067&rft.eissn=1422-0067&rft_id=info:doi/10.3390/ijms24119641&rft_dat=%3Cgale_pubme%3EA752743779%3C/gale_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2824046980&rft_id=info:pmid/37298590&rft_galeid=A752743779&rfr_iscdi=true |