Abstract
Ceramide and diacylglycerol (DAG) are bioactive lipids and mediate many cellular signaling pathways. Sphingomyelin synthase (SMS) is the single metabolic link between the two, while SMS2 is the only SMS form located at the plasma membrane. SMS2 functions were investigated in HepG2 cell lines stably expressing SMS2. SMS2 overexpression did not alter sphingomyelin (SM), phosphatidylcholine (PC), or ceramide levels. DAG content increased by approx. 40% and led to downregulation of DAG-dependent protein kinase C (PKC). SMS2 overexpression also induced senescence, characterized by positivity for β-galactosidase activity and heterochromatin foci. HepG2-SMS2 cells exhibited protruded mitochondria and suppressed mitochondrial respiration rates. ATP production and the abundance of Complex V were substantially lower in HepG2-SMS2 cells as compared to controls. SMS2 overexpression was associated with inflammasome activation based on increases in IL-1β and nlpr3 mRNA levels. HepG2-SMS2 cells exhibited lipid droplet accumulation, constitutive activation of AMPK based on elevated 172Thr phosphorylation, increased AMPK abundance, and insensitivity to insulin suppression of AMPK. Thus, our results show that SMS2 regulates DAG homeostasis and signaling in hepatocytes and also provide proof of principle for the concept that offset in bioactive lipids’ production at the plasma membrane can drive the senescence program in association with steatosis and, seemingly, by cell-autonomous mechanisms.
Highlights
Ceramide and diacylglycerol (DAG) are the archetypical lipid second messengers that mediate cellular responses to growth factors, cytokines, and other extracellular signals.In a healthy state, the generation and turnover of these two bioactive lipids are strictly regulated to ensure a controlled, transient rise in their local concentrations followed by the activation of downstream signaling molecules such as protein kinase C (PKC) [1], which is regulated by DAG, or protein phosphatase 2A (PP2A) [2] and PKCζ [3], which are among the few identified direct ceramide targets
The generation and turnover of these two bioactive lipids are strictly regulated to ensure a controlled, transient rise in their local concentrations followed by the activation of downstream signaling molecules such as protein kinase C (PKC) [1], which is regulated by DAG, or protein phosphatase 2A (PP2A) [2] and PKCζ [3], which are among the few identified direct ceramide targets
Single cell-derived colonies were expanded under G418 selective pressure and are referred to as HepG2-SMS2 or HepG2-empty vector (EV), respectively
Summary
Ceramide and diacylglycerol (DAG) are the archetypical lipid second messengers that mediate cellular responses to growth factors, cytokines, and other extracellular signals. The generation and turnover of these two bioactive lipids are strictly regulated to ensure a controlled, transient rise in their local concentrations followed by the activation of downstream signaling molecules such as protein kinase C (PKC) [1], which is regulated by DAG, or protein phosphatase 2A (PP2A) [2] and PKCζ [3], which are among the few identified direct ceramide targets. DAG is involved in pro-survival, proliferative, and metabolic signals, while ceramide participates in the cellular response to stress and in the onset of cell death. SMS transfers the phosphorylcholine group from phosphatidylcholine (PC) to ceramide, producing SM and DAG. A third form, SMSr, exists in the endoplasmic reticulum and catalyzes the synthesis of ceramide phosphoethanolamine, a compound that can be converted to SM by a three-step methylation reaction at the primary amine of the ethanolamine head group
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