Regulation of ceramide-1-phosphate metabolism in cell signaling networks

University Of Virginia, Charlottesville VA

Investigators

Abstract

Metabolic reprogramming (MR) is an important cellular mechanism in response to cell stress & agonist-induced proliferation/polarization, which is characterized by the suppression of oxidative phosphorylation (OXPHOS) & subsequent upregulation of aerobic glycolysis, lipid metabolism, mitochondrial fission, and the oxidative pentose phosphate pathway (PPP). The key signals that regulate and induce MR are currently unclear, but our laboratory found that ceramide-1-phosphate (C1P), a bioactive sphingolipid produced by the phosphorylation of ceramide by ceramide kinase (CERK), was required for this type of MR & for the induction of oxidative DE-phosphorylation (dePhos), a novel ATP-backflow mechanism that maintains mitochondrial membrane potential (MMP). Specifically, CERK ablation blocked the MR of macrophages (MFs) to aerobic glycolysis induced by lipopolysaccharide (LPS) & interferong (IFNg), which also inhibited M1 polarization & induced ferroptotic cell death. This finding translated in vivo as CERK ablation significantly reduced M1 MFs in inflammatory mouse models. Mechanistic studies showed that initiation of M1/N1 polarization of innate immune cells induced cardiolipin (CL)-dependent recruitment of CERK to the mitochondria & significant levels of mitochondrial C1P (Mito-C1P). Mito-C1P induction was also completely abrogated by CERK ablation, which destabilized the interaction of the voltage dependent anion channel (VDAC) with free tubulin, increased reactive oxygen species (ROS), and induced hyperpolarization of the MMP. CERK-derived C1P also regulates signaling pathways linked to mitofission & lipid droplet (LD) biosynthesis, the latter via the induction of the direct C1P target, cPLA2a. LD biogenesis is a key step in ferroptosis protection for cells undergoing MR by sequestering/esterifying polyunsaturated fatty acids (PUFAs) as these lipids are sensitive to the increased ROS levels linked to MR. In this regard, initial data also show increased levels of LDs during M1 MF polarization, which was negated by CERK ablation & loss of the C1P:cPLA2a interaction. Therefore, we hypothesize that CERK is a previously undefined molecular “rheostat” for MR acting on several key points in the pathway. This hypothesis will be interrogated by three specific aims: 1) Determine the role of CERK-derived C1P in metabolic programming; 2) Determine the mechanism of CERK activation in metabolic reprogramming; & 3) Determine the C1P targets regulating metabolic reprogramming. Our innovation & significance are at least three-fold: 1) the delineation of a novel “rheostat”/“switch” for MR, which may have unifying themes across biological models & disease states; 2) the demonstration of sphingolipid topology (e.g., mitochondria) linked to a specific signaling paradigm, which is a current scientific gap for sphingolipid second messengers; and 3) the characterization of the role of a C1P interacting factor in MR.