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Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake.

Ahn KH, Kim SK, Choi JM, Jung SY, Won JH, Back MJ, Fu Z, Jang JM, Ha HC, Kim DK - PLoS ONE (2013)

Bottom Line: Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells.Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase.Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, Dongjak-Ku, Seoul, South Korea.

ABSTRACT
Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH-optimum and Mg(2+)-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

No MeSH data available.


Related in: MedlinePlus

Effect of Hsp60 siRNA on N-SMase activity, ceramide production, and dopamine re-uptake in PC12 cells.PC12 cells were seeded into six-well dishes and transfected with the negative control (scramble) or Hsp60 siRNA (20 nM). After 48 h, the cells lysed, and N-SMase activity was determined. The results represent the mean ± S.D. of seven independent experiments (A). After 48 h transfection, total RNA was isolated and purified from the PC12 cells. Real-time PCR was performed using primers specific for N-SMase2 and β-actin (the values are normalized to β-actin) (B). After 24 h transfection, the medium was changed, and cells were labeled with [3H] palmitic acid. Total lipids were extracted from the PC12 cells and separated with radioactive ceramide using thin-layer chromatography. The ceramide levels in the cells were measured as described under “Experimental Procedures.” (C). Scramble, N-SMase2, and Hsp60 siRNA were transfected into PC12 cells, and 48 h later, fresh medium was added with [3H] DA. The medium was removed, and the cells were washed. PC12 cells were treated with A23187, and the culture was continued for the indicated time periods. An aliquot of each culture's supernatants were taken, and the radioactivity was measured (E). After 48 h transfection, fresh medium was added with [3H] DA. DA uptake was measured after 2 h incubation at 37°C. The results represent the mean ± S.D. of three independent experiments (F). *p<0.05 compared with scramble control.
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pone-0067216-g009: Effect of Hsp60 siRNA on N-SMase activity, ceramide production, and dopamine re-uptake in PC12 cells.PC12 cells were seeded into six-well dishes and transfected with the negative control (scramble) or Hsp60 siRNA (20 nM). After 48 h, the cells lysed, and N-SMase activity was determined. The results represent the mean ± S.D. of seven independent experiments (A). After 48 h transfection, total RNA was isolated and purified from the PC12 cells. Real-time PCR was performed using primers specific for N-SMase2 and β-actin (the values are normalized to β-actin) (B). After 24 h transfection, the medium was changed, and cells were labeled with [3H] palmitic acid. Total lipids were extracted from the PC12 cells and separated with radioactive ceramide using thin-layer chromatography. The ceramide levels in the cells were measured as described under “Experimental Procedures.” (C). Scramble, N-SMase2, and Hsp60 siRNA were transfected into PC12 cells, and 48 h later, fresh medium was added with [3H] DA. The medium was removed, and the cells were washed. PC12 cells were treated with A23187, and the culture was continued for the indicated time periods. An aliquot of each culture's supernatants were taken, and the radioactivity was measured (E). After 48 h transfection, fresh medium was added with [3H] DA. DA uptake was measured after 2 h incubation at 37°C. The results represent the mean ± S.D. of three independent experiments (F). *p<0.05 compared with scramble control.

Mentions: Next, we carried out experiments to determine whether Hsp60 affects N-SMase activity and ceramide production. PC12 cells were transfected with scramble or Hsp60 siRNA and were subsequently analyzed for N-SMase activity, N-SMase2 mRNA levels, and ceramide levels. Hsp60 knockdown effectively increased Mg2+-dependent N-SMase activity (∼25%; Figure 9A) and ceramide production (∼20%; Figure 9C). Additionally, we determined the levels of various endogenous ceramides. C16- and C24-ceramide levels were increased significantly by knockdown Hsp60 (Figure 9D). However, treatment with Hsp60 siRNA did not change N-SMase2 mRNA levels (Figure 9B). Hence, Hsp60 knockdown increased N-SMase activity and ceramide levels without induction of N-SMase2 mRNA.


Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake.

Ahn KH, Kim SK, Choi JM, Jung SY, Won JH, Back MJ, Fu Z, Jang JM, Ha HC, Kim DK - PLoS ONE (2013)

Effect of Hsp60 siRNA on N-SMase activity, ceramide production, and dopamine re-uptake in PC12 cells.PC12 cells were seeded into six-well dishes and transfected with the negative control (scramble) or Hsp60 siRNA (20 nM). After 48 h, the cells lysed, and N-SMase activity was determined. The results represent the mean ± S.D. of seven independent experiments (A). After 48 h transfection, total RNA was isolated and purified from the PC12 cells. Real-time PCR was performed using primers specific for N-SMase2 and β-actin (the values are normalized to β-actin) (B). After 24 h transfection, the medium was changed, and cells were labeled with [3H] palmitic acid. Total lipids were extracted from the PC12 cells and separated with radioactive ceramide using thin-layer chromatography. The ceramide levels in the cells were measured as described under “Experimental Procedures.” (C). Scramble, N-SMase2, and Hsp60 siRNA were transfected into PC12 cells, and 48 h later, fresh medium was added with [3H] DA. The medium was removed, and the cells were washed. PC12 cells were treated with A23187, and the culture was continued for the indicated time periods. An aliquot of each culture's supernatants were taken, and the radioactivity was measured (E). After 48 h transfection, fresh medium was added with [3H] DA. DA uptake was measured after 2 h incubation at 37°C. The results represent the mean ± S.D. of three independent experiments (F). *p<0.05 compared with scramble control.
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pone-0067216-g009: Effect of Hsp60 siRNA on N-SMase activity, ceramide production, and dopamine re-uptake in PC12 cells.PC12 cells were seeded into six-well dishes and transfected with the negative control (scramble) or Hsp60 siRNA (20 nM). After 48 h, the cells lysed, and N-SMase activity was determined. The results represent the mean ± S.D. of seven independent experiments (A). After 48 h transfection, total RNA was isolated and purified from the PC12 cells. Real-time PCR was performed using primers specific for N-SMase2 and β-actin (the values are normalized to β-actin) (B). After 24 h transfection, the medium was changed, and cells were labeled with [3H] palmitic acid. Total lipids were extracted from the PC12 cells and separated with radioactive ceramide using thin-layer chromatography. The ceramide levels in the cells were measured as described under “Experimental Procedures.” (C). Scramble, N-SMase2, and Hsp60 siRNA were transfected into PC12 cells, and 48 h later, fresh medium was added with [3H] DA. The medium was removed, and the cells were washed. PC12 cells were treated with A23187, and the culture was continued for the indicated time periods. An aliquot of each culture's supernatants were taken, and the radioactivity was measured (E). After 48 h transfection, fresh medium was added with [3H] DA. DA uptake was measured after 2 h incubation at 37°C. The results represent the mean ± S.D. of three independent experiments (F). *p<0.05 compared with scramble control.
Mentions: Next, we carried out experiments to determine whether Hsp60 affects N-SMase activity and ceramide production. PC12 cells were transfected with scramble or Hsp60 siRNA and were subsequently analyzed for N-SMase activity, N-SMase2 mRNA levels, and ceramide levels. Hsp60 knockdown effectively increased Mg2+-dependent N-SMase activity (∼25%; Figure 9A) and ceramide production (∼20%; Figure 9C). Additionally, we determined the levels of various endogenous ceramides. C16- and C24-ceramide levels were increased significantly by knockdown Hsp60 (Figure 9D). However, treatment with Hsp60 siRNA did not change N-SMase2 mRNA levels (Figure 9B). Hence, Hsp60 knockdown increased N-SMase activity and ceramide levels without induction of N-SMase2 mRNA.

Bottom Line: Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells.Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase.Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, Dongjak-Ku, Seoul, South Korea.

ABSTRACT
Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH-optimum and Mg(2+)-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

No MeSH data available.


Related in: MedlinePlus