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.

Keywords: Clathrin-mediated endocytosis; Hsc70; Reactive oxygen species; Sp1; Very long-chain ceramides.

Fig. 1.

Characterization of CerS2 null astrocytes. (A) Astrocytes were purified and co-stained with antibodies against GFAP (green) and CD68 (red). Nuclei were stained with Hoechst 33348 (blue). Scale bar: 40 μm. (B) mRNA levels of CerS isoforms, normalized to expression of HPRT. Values are means±s.e.m., n≥3. ***P<0.001 (Student's t-test). (C) Activity of CerS proteins using different acyl-CoAs. Values are means±s.e.m., n≥4. ***P<0.001 (Student's t-test). (D) Mass spectrometry analysis of ceramide species. Values are means±s.e.m., n=3 for CerS2 null, n=2 for WT. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test). The inset shows total ceramide levels. In panels B-E, WT data is black and CerS2 null is gray.

Fig. 2.

The rate of CME in CerS2 null astrocytes. Cells were incubated with (A) Dil–LDL, (B) Cy5–Tf or (C) BODIPY–LacCer for various times, and the amount of internalized ligand was analyzed. Values are means±s.e.m., n=3. *P<0.05; **P<0.01 (Student's t-test). WT is indicated by circles and CerS2 null by squares.

Fig. 3.

Dysregulated CME in CerS2 null astrocytes. (A) Western blot of clathrin heavy chain (CHC; using a pan-CHC antibody), LDL receptor (LDL-R) and Tf receptor (Tf-R) in astrocytes. GAPDH was used as a loading control. The right-hand panel shows ratio values calculated from quantification of the western blots. Values are means±s.e.m. **P<0.01 (Student's t-test). (B) Distribution of CHC determined by confocal microscopy. CHC, red; Hoechst 33348, blue. Arrowheads indicate large puncta of CHC in CerS2 null astrocytes. The images are representative of three independent experiments. Scale bars: 10 μm. (C) mRNA levels of auxilin-1 and Hsc70, normalized to expression of TBP. n=2. Values are means±s.d. *P<0.05 (Student's t-test). (D) Western blot of Hsc70. GAPDH was used as a loading control. The lower panel shows ratio values calculated by quantifying the western blots. Values are means±s.e.m., n=5. **P<0.01.

Fig. 4.

Sp1 regulates Hsc70 in CerS2 null astrocytes. (A) Western blot of Sp1 and HSF-1 in WT and CerS2 null cells. GAPDH was used as a loading control. The right-hand panel shows ratio values calculated from quantification of the blots. Values are means±s.d., n=4. *P<0.05 (Student's t-test). (B) mRNA levels of Sp1, normalized to expression of TBP. Values are means±s.d., n=4. (C) Schematic representation of the promoter region of the mouse Hsc70 (Hspa8) gene. The first exon is shown as a gray box. The transcription start site (TSS) and the two putative Sp1-binding sites are also indicated. Schematic based on mouse genome version GRCm38, chr9:40,801,066-40,801,348. (D) ChIP analysis of the binding of Sp1 transcription factor to positions −68 and −174 in the Hsc70 promoter. An antibody against HA was used as a binding control for Sp1 binding. n=3, values are means±s.e.m. *P<0.05 (Student's t-test). (E) mRNA levels of fatty acid synthase, glycolipid transfer protein, toll-like receptor 4 (TLR4) and GFAP. Values are means± s.e.m., n=3. *P<0.05; ***P<0.001 (Student's t-test).

Fig. 5.

Increased oxidative stress in CerS2 null astrocytes. (A) FACS analysis of ROS levels in WT (green) and CerS2 null (red) astrocytes using cmH2DCF-DA. Individual samples contained at least 30,000 cells. The right-hand panel shows quantification of ROS in CerS2 null cells relative to those in WT. n=3. Values are means±s.e.m. ***P<0.001 (Student's t-test). (B) Western blot of 3-nitrotyrosine. GAPDH was used as a loading control. The right-hand panel shows quantification. n=4. Values are means±s.d., **P<0.01 (Student's t-test). (C) Activity of mitochondrial complex IV normalized to that of citrate synthase (CS) in WT and CerS2 null astrocytes. Values are means±s.d., n=4. ***P<0.001 (Student's t-test).

Fig. 6.

Oxidative stress regulates CME through the Sp1–Hsc70 axis. Astrocytes were incubated with and without H₂O₂ (100 μM, 4 h) or NAC (100 μM, 4 h), followed by incubation with (A) Cy5–Tf (red) or (C) Dil–LDL (red) for the indicated times. Hoechst 33348, blue. Scale bars: 10 μm. Rate of uptake of (B) Cy5–Tf or (D) Dil–LDL was measured. Values are means±s.e.m., n=3. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test). (E) Western blot of Sp1, Hsc70, transferrin receptor and LDL receptor. GAPDH was used as a loading control. The right-hand panel shows ratio values calculated from quantification of western blots. Values are means±s.e.m., n=3. *P<0.05; **P<0.01 (Student's t-test).

Fig. 7.

Putative scheme of the effect of altering the SL acyl chain length on CME. Altering the SL acyl chain length (red) can either lead to changes in membrane biophysical properties or to activation of downstream pathways (blue). In the current study (black), we demonstrate the activation of one such pathway, which eventually leads to a reduced rate of CME.