Oxidative stress elicited by modifying the ceramide acyl chain length reduces the rate of clathrin-mediated endocytosis

Abstract

Sphingolipids modulate clathrin-mediated endocytosis (CME) by altering the biophysical properties of membranes. We now examine CME in astrocytes cultured from ceramide synthase 2 (CerS2) null mice, which have an altered sphingolipid acyl chain composition. The rate of endocytosis of low-density lipoprotein and transferrin, which are internalized via CME, was reduced in CerS2 null astrocytes, although the rate of caveolin-mediated endocytosis was unaltered. Levels of clathrin heavy chain were increased, which was due to decreased levels of Hsc70 (also known as HSPA8), a protein involved in clathrin uncoating. Hsc70 levels were decreased because of lower levels of binding of Sp1 to position −68 in the Hsc70 promoter. Levels of Sp1 were downregulated due to oxidative stress, which was elevated fourfold in CerS2 null astrocytes. Furthermore, induction of oxidative stress in wild-type astrocytes decreased the rate of CME, whereas amelioration of oxidative stress in CerS2 null astrocytes reversed the decrease. Our data are consistent with the notion that sphingolipids not only change membrane biophysical properties but also that changes in their composition can result in downstream effects that indirectly impinge upon a number of cellular pathways, such as CME.

Agonists that stimulate secretion promote the recruitment of CFTR into membrane lipid microdomains

“…The latter might be explained by spatial isolation of CFTR in platforms from endocytosis machinery or reduced membrane invagination due to the rigidity of tightly packed ceramide acyl chains in platforms (Pinto et al, 2013). Ceramides are implicated in endocytosis as ceramide synthase (Cers) 2–null mice have reduced clathrin–mediated endocytosis, an effect that can be explained by a fourfold increase in reactive oxygen species production when very long chain ceramide (VLCC) levels are reduced (Volpert et al, 2017). In addition to increasing CFTR abundance at the cell surface, secretagogue-induced partitioning into microdomains may modulate its regulation and the pharmacological rescue of mutant CFTR by corrector drugs used to treat CF.…”

Endocytosis in the adaptation to cellular stress

“…For example, elevated ROS were shown to inhibit EGFR and lectin receptor endocytosis in fibroblasts, likely by preventing their ubiquitination [ 90 ]. Similarly, ROS were observed to impair the internalization of GLUT4 in cardiomyocytes [ 91 ], aquaporin 4 (AQP4), transferrin and LDL in astrocytes [ 92 , 93 ], and of synaptic vesicle membranes in neurons [ 94 ].…”

“…Regarding the potential underlying mechanism, oxidative conditions in astrocytes were reported to lead to the downregulation of the transcription factor Sp1, which promotes the transcription of Hsc70 [ 93 ], a constitutively expressed heat shock protein and an essential cofactor for the uncoating of Clathrin-coated vesicles in CME [ 24 ]. Decreased levels of Hsc70 are expected to reduce the availability of free Clathrin for new rounds of endocytosis, since it would be sequestered in uncoating-deficient Clathrin-coated vesicles or Clathrin cages [ 93 ]. However, it is unclear whether this mechanism extends to other cell types and conditions.…”

Orm/ORMDL proteins: Gate guardians and master regulators

“…As outlined in the other articles in this issue, sphingolipids are a diverse family of lipids with an enormous impact on cell and organismal biology. Clearly, the regulation of the production of these lipids is essential for the proper functioning of cells and tissues, and dysregulation of their synthesis can have catastrophic effects (Harrison, Dunn, and Campopiano 2018; Kolter and Sandhoff 2006; Marcus et al 2006; Maceyka and Spiegel 2014; Obeid et al 1993; Miller et al 2014; Sasset et al 2016; Volpert et al 2017; Ogretmen 2018; Olsen and Faergeman 2017; Espaillat, Kew, and Obeid 2017; Shamseddine, Airola, and Hannun 2015).…”