Inhibition of sphingomyelin synthase (SMS) affects intracellular sphingomyelin accumulation and plasma membrane lipid organization

Abstract

Sphingomyelin plays a very important role both in cell membrane formation that may well have an impact on the development of diseases like atherosclerosis and diabetes. However, the molecular mechanism that governs intracellular and plasma membrane SM levels is largely unknown. Recently, two isoforms of sphingomyelin synthase (SMS1 and SMS2), the last enzyme for SM de novo synthesis, have been cloned. We have hypothesized that SMS1 and SMS2 are the two most likely candidates responsible for the SM levels in the cells and on the plasma membrane. To test this hypothesis, cultured cells were treated with tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of SMS, or with SMS1 and SMS2 siRNAs. Cells were then pulsed with [14C]-L-serine (a precursor of all sphingolipids). SMS activity and [14C]-SM in the cells were monitored. We found that SMS activity was significantly decreased in cells after D609 or SMS siRNA treatment, compared with controls. SMS inhibition by D609 or SMS siRNAs significantly decreased intracellular [14C]-SM levels. We measured cellular lipid levels, including SM, ceramide, phosphatidylcholine, and diacylglycerol and found that SMS1 and SMS2 siRNA treatment caused a significant decrease of SM levels (20% and 11%, respectively), compared to control siRNA treatment; SMS1 but not SMS2 siRNA treatment caused a significant increase of ceramide levels (10%). There was a decreasing tendency for diacylglycerol levels after both SMS1 and SMS2 siRNA treatment, however, it was not statistical significant. As shown by lipid rafts isolation and lipid determination, SMS1 and SMS2 siRNA treatment led to a decrease of SM content in detergent-resistant lipid rafts on the cell membrane. Furthermore, SMS1 and SMS2 siRNA-treated cells had a stronger resistance than did control siRNA-treated cells to lysenin (a protein that causes cell lysis due to its affinity for plasma membrane SM). These results indicate that both SMS1 and SMS2 contribute to SM de novo synthesis and control SM levels in the cells and on the cell membrane including plasma membrane, implying an important relationship between SMS activity and cell functions.

FIG. 1. D609 treatment caused decrease of SMS activity and decrease of intracellular and secreted SM levels in cells

Two doses of D609 (300 μM and 600 μM) were added to Huh7 cell (A, B), HepG2 cell (C, D), HEK 293 cell (E, F) and CHO cell (G, H) culture medium, together with 0.2 mM oleic acid and 0.2 μci/ml of [¹⁴C]-L-serine. After 24 h of incubation, cells were harvested. Lipids were extracted and intracellular [¹⁴C]-SM levels were quantitated as described in “Experimental procedure.” A, C, E, and G, Quantitative display of SMS activity. The reaction system contained 50 mM Tris-HCl (pH 7.4), 25 mM KCl, C₆-NBD-ceramide (0.1 μg/μl), and phosphotidylcholine (0.01 μg/μl). The mixture was incubated at 37°C for 2 hours. Lipids were extracted in chloroform: methanol (2:1), dried under N₂ gas, and separated by thin layer chromatography (TLC) using Chloroform:MeOH:NH₄OH (14:6:1). B, D, F, and H, quantitative displays of intracellular [¹⁴C]-SM levels. Values are mean ± S.D., n = 5, p < 0.001 by ANOVA. Columns labeled with different lower-case letters (a-c) are statistically different by SNK test (p < 0.05).

FIG. 2. siRNA treatment decreased SMS1 and SMS2 mRNA levels in Huh7 cells

SMS1 and SMS2 siRNAs were utilized to transfect Huh7 cells. After 24 h of transfection, total RNA was extracted from the cells. A, SMS1 mRNA in Huh7 cells was measured by quantitative real-time PCR. B, SMS2 mRNA in Huh7 cells was measured by quantitative real-time PCR. Expression was described as the ratio of SMS1 or SMS2 mRNA to 18S rRNA. Values are mean ± S.D., n = 3, p < 0.001 by ANOVA. Columns labeled with different lower-case letters (a-c) are statistically different by SNK test (p < 0.0001). siR1.1, SMS1 siRNA1; siR1.2, SMS1 siRNA2; siR2.1, SMS2 siRNA1; siR2.2, SMS2 siRNA2.

FIG. 3. . The effect of SMS1 and SMS2 siRNAs on SMS activity and intracellular [¹⁴C]-SM levels

SMS1 and SMS2, or SMS1 plus SMS2 siRNAs, were utilized to transfect Huh7 (A, B) and HEK 293 (C, D) cells. After 24 h of transfection, 0.2 mM oleic acid and 0.2 μci/ml of [¹⁴C]-L-serine were added to the cell culture medium. Intracellular [¹⁴C]-SM levels were quantitated as described in “Experimental procedure.” A and C, quantitative display of SMS activity. B and D, quantitative displays of intracellular [¹⁴C]-SM levels. Values are mean ± S.D., n=5, p < 0.01 by ANOVA. Columns labeled with different lower-case letters are statistically different by SNK test (p < 0.05).

FIG. 4. A time course of SMS1 and SMS2 siRNAs on intracellular [¹⁴C]-SM levels

SMS1 and SMS2 siRNAs were utilized to transfect Huh7 cells. After 24 h of transfection, 0.2 mM oleic acid and 0.2 μci/ml of [¹⁴C]-L-serine were added to the cell culture medium. Intracellular [¹⁴C]-SM levels were quantitated as described in “Experimental procedure” after 1 and 2 hour incubation. Values are mean ± S.D., n=3, p < 0.01 by ANOVA. Columns labeled with different lower-case letters are statistically different by SNK test (p < 0.05).

FIG. 5. Isolation of lipid rafts and non-rafts region from HEK 293 cells

SMS1 and SMS2, or combined siRNAs were utilized to transfect HEK 293 cells. After 48 h of transfection, detergent insoluble and soluble membrane domains were separated by sucrose gradients. Fractions (–9) were collected from the top of the gradient. Each fraction (100 μg protein) was used for Western blot for Lyn and CD71. SM and cholesterol in each fraction were determined by enzymatic assays. A, Western blot for Lyn and CD71 on lipid raft and non-raft regions. B, SM measurement in fractions. C, cholesterol measurement in fractions. Values are mean ± S.D., n = 4, * p < 0.001 by ANOVA. For SM measurement: control vs. siR1, p < 0.01, control vs. siR2, p < 0.05, and control vs. siR1/siR2, p < 0.01 in fractions 3 and 4, respectively. siR1 and siR2, siRNAs for SMS1 and SMS2, respectively; siR1/R2, siRNAs for SMS1 plus SMS2.

FIG. 6. SMS1 and SMS2 gene knockdown decreased lysenin-mediated cell mortality

A, SMS1 and SMS2, or combined siRNAs, were utilized to transfect Huh7 cells. After 24 h of transfection, lysenin (200 ng/ml) was added to the cell culture medium and cell mortality was monitored by WST-1 Cell Proliferation Reagent (Roche). B, SMS1 and SMS2, or siRNAs were utilized to transfect HEK 293 cells. The rest was same as the Huh7 cell experiment. Values are mean ± S.D., n = 5, p < 0.001 by ANOVA. Columns labeled with different lower-case letters are statistically different by Student t test (p < 0.05). siR1 and siR2, siRNAs for SMS1 and SMS2, respectively; siR1/R2, siRNAs for SMS1 plus SMS2.