TY - JOUR
T1 - Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages
AU - Muller, Ittai B.
AU - Lin, Marry
AU - Jonge, Robert de
AU - Will, Nico
AU - López-Navarro, Baltasar
AU - Laken, Conny van der
AU - Struys, Eduard A.
AU - Oudejans, Cees B. M.
AU - Assaraf, Yehuda G.
AU - Cloos, Jacqueline
AU - Puig-Kröger, Amaya
AU - Jansen, Gerrit
N1 - Funding Information: This study was supported in part by a grant (Noyons 2018) from the Dutch Association for Clinical Chemistry (NVKC) to I.B.M. and R.d.J. Additionally, this work was supported by Grant PI20/00316 and Red de Enfermedades Inflamatorias (RICORS RD21/0002/0034) from Instituto de Salud Carlos III, and cofinanced by the European Regional Development Fund “A way to achieve Europe” (ERDF) to A.P.-K. Publisher Copyright: © 2023 by the authors.
PY - 2023/6/1
Y1 - 2023/6/1
N2 - 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.
AB - 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.
KW - alternative splicing
KW - folate metabolism
KW - folylpolyglutamate synthetase
KW - gene expression
KW - macrophages
KW - methotrexate
UR - http://www.scopus.com/inward/record.url?scp=85161722524&partnerID=8YFLogxK
U2 - https://doi.org/10.3390/ijms24119641
DO - https://doi.org/10.3390/ijms24119641
M3 - Article
C2 - 37298590
SN - 1661-6596
VL - 24
JO - International journal of molecular sciences
JF - International journal of molecular sciences
IS - 11
M1 - 9641
ER -