Lactosylceramide‐Induced Phosphorylation Signaling to Group IVA Phospholipase A₂ via Reactive Oxygen Species in Tumor Necrosis Factor‐α‐Treated Cells

Abstract

The activity of α-type cytosolic phospholipase A (cPLA α, group IVA PLA ), which releases arachidonic acid (AA), is mainly regulated by the Ca -induced intracellular translocation/attachment of the enzyme to substrate membranes and its phosphorylation. We previously reported that tumor necrosis factor-α (TNFα) stimulated the formation of lactosylceramide (LacCer) in L929 fibroblast cells, and this lipid directly bound with and activated cPLA α [Nakamura et al. [2013] J. Biol. Chem. 288:23264-23272]. We herein investigated the role of phosphorylation signaling in the TNFα/LacCer-induced activation of cPLA α in cells. TNFα-treated L929 cells released AA via the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cPLA α, while a treatment with LacCer alone released AA in a similar manner. The TNFα-induced responses including release of AA were decreased by the inhibition of LacCer synthesis. The treatment with TNFα and LacCer increased the levels of reactive oxygen species (ROS), and the reduction/scavenging of ROS decreased the phosphorylation cascade and release of AA in TNFα/LacCer-treated L929 cells. In the cell line CHO, the treatment with LacCer stimulated the phosphorylation cascade and release of AA via the formation of ROS. Treatments with the anti-LacCer antibody and 4β-phorbol 12-myristate 13-acetate stimulated the phosphorylation cascade, but did not release AA by itself. When combined with the Ca ionophore A23187, treatments with the anti-LacCer antibody and 4β-phorbol 12-myristate 13-acetate released AA. These results, including our previous findings, showed that LacCer alone simultaneously stimulates two processes to activate cPLA α: a phosphorylation signal and attachment of the enzyme to substrate membranes. J. Cell. Biochem. 118: 4370-4382, 2017. © 2017 Wiley Periodicals, Inc.

Metabolomics as a Promising Resource Identifying Potential Biomarkers for Inflammatory Bowel Disease

“…In fact, neutrophils show high levels of lactosylceramide and the proinflammatory cytokine TNF-α favours, in turn, the lactosylceramide production [68]. In addition, lactosylceramide is also responsible of stimulating phospholipase A2 and arachidonic acid release [69], a lipid mediator in inflammatory disease as previously explained. Therefore, it is clear that lactosylceramide might represent a promising biomarker for IBD patients, which needs to be validated in bigger cohorts of IBD patients and better studied in order to characterize its specific role in IBD.…”

Convergence: Lactosylceramide-Centric Signaling Pathways Induce Inflammation, Oxidative Stress, and Other Phenotypic Outcomes

“…In mouse L cells, treatment with TNF-α led to the generation of LacCer, the translocation of cPLA2 from the cytosol to the Golgi apparatus (a Ca 2+ -independent process/activation), and the release of arachidonic acid [110]. In another study, mouse fibroblasts cells treated with LacCer alone could simultaneously initiate phosphorylation of cPLA2α via the PKC/MEK/ERK-dependent pathway at the plasma membrane as well as the attachment of cPLA2α to substrate membranes such as the Golgi complex [109]. Similarly, in the CHO cell line, LacCer treatment resulted in a phosphorylation cascade and release of arachidonic acid [110].…”

“…However, cPLA2 activation and not ROS generation is the upstream signaling molecule for CD11B expression, followed by neutrophil adhesion to ICAM-1 expressed in endothelial cells [82]. The activity of cPLA2α increases in response to high intracellular free calcium concentrations and phosphorylation by several kinases including ERK1/2 [109]. The binding of calcium to the C2 domain of cPLA2α stimulates the translocation of the enzyme to areas surrounding the nucleus.…”

“…However, recent studies show that certain lipids such as ceramide-1-phosphate and LacCer can activate cPLA2α by stimulating or promoting its attachment to substrate membranes in a calcium-independent manner [109]. In mouse L cells, treatment with TNF-α led to the generation of LacCer, the translocation of cPLA2 from the cytosol to the Golgi apparatus (a Ca 2+ -independent process/activation), and the release of arachidonic acid [110].…”

Intersections between copper, β-arrestin-1, calcium, FBXW7, CD17, insulin resistance and atherogenicity mediate depression and anxiety due to type 2 diabetes mellitus: a nomothetic network approach

“…Our findings may be explained by the effects of LacCer on pattern recognition receptors leading to activated immune, oxidative and nitrosative pathways [40-43]. Moreover, LacCer is a direct cytosolic phospholipase alpha 2 activator (cPLA2α) by stimulating phosphorylation signals and attachment of the enzyme to substrate membranes [80], a phenomenon that is associated with activated inflammatory pathways. Furthermore, increased levels of serum LacCer predict cardiovascular disease progression and mortality above and beyond the effects of established risk factors [81].…”