Single-Cell Transcriptome Dynamics of the Autotaxin-Lysophosphatidic Acid Axis During Muscle Regeneration Reveal Proliferative Effects in Mesenchymal Fibro-Adipogenic Progenitors

Lysophosphatidic acid (LPA) is a growth factor-like bioactive phospholipid recognising LPA receptors (LPARs) and mediating signalling pathways that regulate embryonic development, wound healing, carcinogenesis, and fibrosis, via effects on cell migration, proliferation and differentiation. Extracellular LPA is generated from lysophospholipids by the secreted hydrolase - ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2; also, AUTOTAXIN / ATX) and metabolised by different membrane-bound phospholipid phosphatases (PLPPs). Here, we use public bulk and singlecell RNA sequencing datasets to explore the gene expression of Lpar 1-6, Enpp2 , and Plpp genes under skeletal muscle homeostasis and regeneration conditions. We show that the skeletal muscle system dynamically expresses the Enpp2-Lpar-Plpp gene axis, with Lpar1 being the highest expressed member among LPARs. Lpar1 was expressed by mesenchymal fibro-adipogenic progenitors (FAPs) and tenocytes, whereas FAPs mainly expressed Enpp2 . Clustering of FAPs identified populations representing distinct cell states with robust Lpar1 and Enpp2 transcriptome signatures in homeostatic cells expressing higher levels of markers Dpp4 and Hsd11b1 . However, tissue injury induced transient repression of Lpar genes and Enpp2 . The role of LPA in modulating the fate and differentiation of tissue-resident FAPs has not yet been explored. Ex vivo, LPAR1/3 and ENPP2 inhibition significantly decreased the cell-cycle activity of FAPs and impaired fibro-adipogenic differentiation, implicating LPA signalling in the modulation of the proliferative and differentiative fate of FAPs. Together, our results demonstrate the importance of the ENPP2-LPAR-PLPP axis in different muscle cell types and FAP lineage populations in homeostasis and injury, paving the way for further research on the role of this signalling pathway in skeletal muscle homeostasis and regeneration, and that of other organs and tissues, in vivo. Summary Our reanalysis of single-cell transcriptomics revealed the involvement and temporally dynamic expression of the ENPP2-LPAR-PLPP axis in response to skeletal muscle regeneration. ### Competing Interest Statement The authors have declared no competing interest. * ASCs : Adipose stromal cells ATX : Autotaxin CTGF/CCN2 : Connective tissue growth factor ENPP2 : Ectonucleotide pyrophosphatase/phosphodiesterase 2 FAPs : Fibro-adipogenic progenitors GPCRs : G protein-coupled receptors LPARs : LPA receptors LPA : Lysophosphatidic acid MSCs : Mesenchymal stromal cells MuSCs : Muscle stem cells PA : Phosphatidic acid PLPPs : Phospholipid phosphatases scRNAseq : Single-cell RNA sequencing