Diacylglycerol kinase-ε is required for the formation of GPI-anchored CD14 and the LPS-induced proinflammatory responses of macrophages

Diacylglycerol kinase-ε (DGKε) is a unique member of the DGK family with strict specificity toward SAG, stearic/palmitic and arachidonic fatty acid-containing DAG, which produces phosphatidic acid used for the synthesis of phosphatidylinositol (PI). PI and its derivatives orchestrate numerous processes. These include the pro-inflammatory signaling of Toll-like receptor 4 (TLR4) and its accessory protein, CD14, which are activated in macrophages by bacterial lipopolysaccharide (LPS). To assess the role of DGKε in LPS-induced responses, we obtained Raw264.7 cells stably depleted of DGKε and subsequently rescued them with DGKε-Myc. The DGKε-depleted cells were also treated with a synthetic GPI precursor. To assess the activity of DGKε and other DGKs in cellular fractions, a fluorescent assay was used, followed by thin-layer chromatography. RT-qPCR, immunoblotting, ELISA, and flow cytometry were used to examine protein abundance, LPS-induced signaling, and cytokine production. Cytokine expression was also analyzed after TLR2 activation. Cells were fractionated with Triton X-100 and X-114 to examine the distribution of GPI-anchored proteins (GPI-APs). Dgke was silenced with siRNA in mouse bone marrow-derived macrophages (BMDM). SAG phosphorylation was markedly decreased in DGKε-depleted Raw264.7 cells, with the activity of other DGKs unaffected. The DGKε depletion abolished the endosomal TLR4 signaling, engaging TRIF and IRF3. The MyD88-dependent signaling pathway was partially inhibited. No mature, GPI-anchored form of CD14 was produced in the DGKε-depleted cells, and residual amounts of CD14 and other GPI-APs were found on the cell surface. The DGKε depletion also inhibited TLR2-mediated cytokine expression and reduced CD14 level in BMDM. The reintroduction of DGKε in Raw264.7 cells restored SAG phosphorylation, total and cell-surface abundance of GPI-CD14 and other GPI-APs, and TLR4 and TLR2 signaling. Treatment of cells with the synthetic GPI precursor partially restored the cell-surface level of CD14. DGKε-dependent phosphorylation of SAG controls the biosynthesis of the GPI moiety of CD14, thereby affecting TLR signaling in macrophages. In addition, DGKε can influence the TLR pathways independently of CD14 formation. Together, these findings identify DGKε as a key factor determining the sensitivity of macrophages to LPS and other microbial components.

Schlagwörter

Fachgebiet (DDC)

570 - Biowissenschaften, Biologie

Projekt

Veranstaltung

Startdatum der Ausstellung

Enddatum der Ausstellung

Startdatum der Konferenz

Enddatum der Konferenz

Datum der letzten Prüfung

ISBN

ISSN

1478-811X

Sprache

Englisch

Während FHNW Zugehörigkeit erstellt

Ja

Zukunftsfelder FHNW

Publikationsstatus

Veröffentlicht

Begutachtung

Peer-Review der ganzen Publikation

Open Access-Status

Gold

Lizenz

Zitation

Hromada‐Judycka, A., Traczyk, G., Amor, I. B., Ciesielska, A., Mąkosa, A., Varon, D., & Kwiatkowska, K. (2026). Diacylglycerol kinase-ε is required for the formation of GPI-anchored CD14 and the LPS-induced proinflammatory responses of macrophages. Cell Communication and Signaling. https://doi.org/10.1186/s12964-026-02884-2

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