Myriocin-mediated up-regulation of hepatocyte apoA-I synthesis is associated with ERK inhibition

Abstract

Sphingolipids including sphingomyelin have been implicated as potential atherogenic lipids. Studies in apoE (apolipoprotein E)-null mice have revealed that the serine palmitoyltransferase inhibitor myriocin reduces plasma levels of sphingomyelin, ceramide, sphingosine-1-phosphate and glycosphingolipids and that this is associated with potent inhibition of atherosclerosis. Interestingly, hepatic apoA-I (apolipoprotein A-I) synthesis and plasma HDL (high-density lipoprotein)-cholesterol levels were also increased in apoE-null mice treated with myriocin. Since myriocin is a known inhibitor of ERK (extracellular-signal-related kinase) phosphorylation, we assessed the possibility that myriocin may be acting to increase hepatic apoA-I production via this pathway. To address this, HepG2 cells and primary mouse hepatocytes were treated with 200 muM myriocin for up to 48 h. Myriocin increased apoA-I mRNA and protein levels by approx. 3- and 2-fold respectively. Myriocin also increased apoA-I secretion up to 3.5-fold and decreased ERK phosphorylation by approx. 70%. Similar findings were obtained when primary hepatocytes were isolated from apoE-null mice that were treated with myriocin (intraperitoneal injection at a dose of 0.3 mg/kg body weight). Further experiments revealed that the MEK (mitogen-activated protein kinase/ERK kinase) inhibitor PD98059 potently inhibited ERK phosphorylation, as expected, and increased primary hepatocyte apoA-I production by 3-fold. These results indicate that ERK phosphorylation plays a role in regulating hepatic apoA-I expression and suggest that the anti-atherogenic mechanism of action for myriocin may be linked to this pathway.

Figure 1. Myriocin up-regulates HepG2 cell apoA-I production

(A) HepG2 cells were treated with 100 or 200 μM myriocin for 24 or 48 h, and mRNA expression was assessed by qPCR. (B and C) HepG2 cells were treated with 100 or 200 μM myriocin for 24 h. The cell medium was collected after 24 h, and apoA-I expression and secretion were analysed by Western blotting. Signal intensity of the apoA-I bands was quantified using Image-J software and the results are expressed relative to controls. Results are means±S.E.M. *P<0.05, **P<0.01 and ***P<0.0001 as assessed by Student's t test.

Figure 2. Myriocin-mediated up-regulation of HepG2 cell apoA-I production is associated with inhibition of ERK phosphorylation

(A) HepG2 cells were treated with 200 μM myriocin for 24 h, and apoA-I, ERK, phospho-ERK (pERK) and β-actin levels were analysed by Western blotting. (B) Signal intensity of the bands quantified by Image-J software. Analysis of apoA-I mRNA expression via qPCR was performed under identical conditions. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. **P<0.01 as assessed by Student's t test.

Figure 3. Myriocin up-regulates mouse hepatocyte apoA-I production

(A) Primary hepatocytes were treated with 200 μM myriocin for 24 h. ApoA-I and β-actin levels were analysed by Western blotting. (B) Signal intensity of the bands was quantified using Image-J software. Analysis of apoA-I mRNA expression via qPCR was performed under identical conditions. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. *P<0.05 and ***P<0.0001 as assessed by Student's t test. cell, cellular, sec, secreted.

Figure 4. Myriocin-mediated up-regulation of mouse hepatocyte apoA-I is associated with inhibition of ERK phosphorylation

(A) Primary mouse hepatocytes were treated with 200 μM myriocin for 24 h. The medium was collected after 24 h. Cellular apoA-I (cell) ERK, phospho-ERK (pERK) and β-actin and secreted apoA-I (sec) levels were analysed by Western blotting. (B) Signal intensity of the bands quantified using Image-J. Analysis of apoA-I mRNA expression via qPCR was performed under identical conditions. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. **P<0.01 and ***P<0.0001 as assessed by Student's t test.

Figure 5. Time course study of myriocin-mediated up-regulation of mouse hepatocyte apoA-I mRNA and its impact on cell viability

(A) Primary mouse hepatocytes were treated with 200 μM myriocin for 0 to 24 h. Cells were extracted in TRIzol® reagent, and apoA-I mRNA expression was assessed by qPCR. (B) Primary mouse hepatocytes were treated with 200 μM myriocin 48 h and cell viability assessed using the MTT reduction assay. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. **P<0.01 and ***P<0.0001 as assessed by Student's t test.

Figure 6. Myriocin decreases SM levels in mouse hepatocytes and HepG2 cells

Cell lysate (5 μl) was added to a 95-μl reaction buffer (see Materials and methods section for details) and, after 45 min of incubation at 37 °C, the absorbance was measured at 595 nm using a spectrophotometric plate reader. The absolute value for total SM, represented by the 100% relative SM, amount for hepatocytes, was 1.20±0.02 nmol/mg of cell protein. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. **P<0.01 and ***P<0.0001 as assessed by Student's t test.

Figure 7. Analysis of apoA-I and ERK levels in hepatocytes derived from myriocin-treated mice

(A) ApoE^−/− mice were injected i.p. with myriocin at 0.3 or 1 mg/kg of body weight or PBS as a vehicle control, as described in the Materials and methods section. The animals were killed, and primary hepatocytes were isolated and assessed for apoA-I, ERK, phospho-ERK (pERK) and β-actin by Western blotting. (B) Signal intensity of the bands was quantified by Image-J software and analysis of apoA-I mRNA expression via qPCR was performed under identical conditions. White bars, control; grey bars, 0.3 mg/kg myriocin-treated; black bars, 1 mg/kg myriocin-treated. Results are means±S.E.M. of three mice in each treatment group. *P<0.05 and **P<0.01 as assessed by Student's t test.

Figure 8. Inhibition of ERK phosphorylation up-regulates mouse hepatocyte apoA-I production

(A) Primary hepatocytes were treated with 50 μM PD98059 for 24 h. The medium was collected after 24 h. Cellular apoA-I (cell), ERK, phospho-ERK (pERK) and β-actin and secreted apoA-I (sec) levels were analysed by Western blotting. (B) Signal intensity of the bands was quantified by Image-J software. Analysis of apoA-I mRNA expression via qPCR was performed under identical conditions. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. *P<0.05, **P< 0.01 and ***P<0.0001 as assessed by Student's t test.