Limits...
Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation.

Kan CF, Singh AB, Stafforini DM, Azhar S, Liu J - J. Lipid Res. (2014)

Bottom Line: AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis.We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination.Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304.

ABSTRACT
ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. Although an association between elevated levels of ACSL4 and hepatosteatosis has been reported, the function of ACSL4 in hepatic FA metabolism and the regulation of its functional expression in the liver remain poorly defined. Here we provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis. The inhibitory action of AA on ACSL4 protein stability could not be prevented by rosiglitazone or inhibitors that interfere with the cellular pathways involved in AA metabolism to biologically active compounds. In contrast, treatment of cells with inhibitors specific for the proteasomal degradation pathway largely prevented the AA-induced ACSL4 degradation. We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination. Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

No MeSH data available.


Related in: MedlinePlus

AA reduces ACSL4 protein half-life without affecting ACSL4 gene transcription or mRNA stability. A: HepG2 cells were treated with 50 μM AA or vehicle for 8 h. CHX, at 5 μg/ml concentration, was added to cells for the indicated times. Total cell lysates were subjected to Western blotting and bands were visualized with antibody against ACSL4 or β-actin. The indicated value is for the blot shown. B: After normalization to β-actin, the ACSL4 signal intensity was plotted against the CHX treatment time to calculate T1/2 of ACSL4 protein. The T1/2 data presented are the mean ± SEM of three independent CHX treatment experiments. C: HepG2 cells were treated with 50 μM AA or vehicle for 8 h. Then, actinomycin D (Act D), at a concentration of 5 μg/ml, was added to the cells and total RNA was isolated at the indicated treatment times for qRT-PCR analysis of ACSL4 and GAPDH. After normalization with GAPDH, ACSL4 mRNA levels were plotted against the treatment time. D: Reporter constructs were cotransfected with a Renilla expression vector (pRL-SV40) into HepG2 cells. Two days post transfection, cell lysates were isolated to measure dual Luc activities. After normalization, the Luc activity of pGL3-basic is expressed as 1 and the Luc activity of pGL3-ACSL4 is expressed as fold of pGL3-basic. E: HepG2 cells were transfected with pGL3-ACSL4 and pRL-SV40 for 2 days prior to AA treatment of 24 h at the indicated doses. Cell lysates were isolated to measure dual Luc activities. The normalized Luc activity in control cells is expressed as 1. F: HepG2 cells were transfected with pcDNA-Luc control vector or pcDNA-Luc-ACSL4 3′UTR plasmid (ACSL4-UTR) for 1 day prior to AA treatment of 24 h at the indicated doses. Cell lysates were isolated to measure dual Luc activities. The normalized Luc activity of pCDNA-Luc in control cells is expressed as 1. n.s., not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109760&req=5

fig4: AA reduces ACSL4 protein half-life without affecting ACSL4 gene transcription or mRNA stability. A: HepG2 cells were treated with 50 μM AA or vehicle for 8 h. CHX, at 5 μg/ml concentration, was added to cells for the indicated times. Total cell lysates were subjected to Western blotting and bands were visualized with antibody against ACSL4 or β-actin. The indicated value is for the blot shown. B: After normalization to β-actin, the ACSL4 signal intensity was plotted against the CHX treatment time to calculate T1/2 of ACSL4 protein. The T1/2 data presented are the mean ± SEM of three independent CHX treatment experiments. C: HepG2 cells were treated with 50 μM AA or vehicle for 8 h. Then, actinomycin D (Act D), at a concentration of 5 μg/ml, was added to the cells and total RNA was isolated at the indicated treatment times for qRT-PCR analysis of ACSL4 and GAPDH. After normalization with GAPDH, ACSL4 mRNA levels were plotted against the treatment time. D: Reporter constructs were cotransfected with a Renilla expression vector (pRL-SV40) into HepG2 cells. Two days post transfection, cell lysates were isolated to measure dual Luc activities. After normalization, the Luc activity of pGL3-basic is expressed as 1 and the Luc activity of pGL3-ACSL4 is expressed as fold of pGL3-basic. E: HepG2 cells were transfected with pGL3-ACSL4 and pRL-SV40 for 2 days prior to AA treatment of 24 h at the indicated doses. Cell lysates were isolated to measure dual Luc activities. The normalized Luc activity in control cells is expressed as 1. F: HepG2 cells were transfected with pcDNA-Luc control vector or pcDNA-Luc-ACSL4 3′UTR plasmid (ACSL4-UTR) for 1 day prior to AA treatment of 24 h at the indicated doses. Cell lysates were isolated to measure dual Luc activities. The normalized Luc activity of pCDNA-Luc in control cells is expressed as 1. n.s., not significant.

Mentions: To determine whether AA lowering of ACSL4 protein levels was due to its increased degradation, we treated HepG2 cells with or without AA and/or with or without a protein synthesis inhibitor, CHX, and changes in ACSL4 protein levels were followed for the next 8 h by the Western blotting. Three separate experiments with identical conditions were performed. Figure 4A is a representative Western blot analysis. Quantitative data presented in Fig. 4B are derived from three independent experiments. AA treatment caused an accelerated degradation of ACSL4 protein with T1/2 = 4.2 ± 0.35 h as compared with T1/2 = 17.3 ± 1.84 h (P < 0.01) calculated from non-AA-treated control cells.


Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation.

Kan CF, Singh AB, Stafforini DM, Azhar S, Liu J - J. Lipid Res. (2014)

AA reduces ACSL4 protein half-life without affecting ACSL4 gene transcription or mRNA stability. A: HepG2 cells were treated with 50 μM AA or vehicle for 8 h. CHX, at 5 μg/ml concentration, was added to cells for the indicated times. Total cell lysates were subjected to Western blotting and bands were visualized with antibody against ACSL4 or β-actin. The indicated value is for the blot shown. B: After normalization to β-actin, the ACSL4 signal intensity was plotted against the CHX treatment time to calculate T1/2 of ACSL4 protein. The T1/2 data presented are the mean ± SEM of three independent CHX treatment experiments. C: HepG2 cells were treated with 50 μM AA or vehicle for 8 h. Then, actinomycin D (Act D), at a concentration of 5 μg/ml, was added to the cells and total RNA was isolated at the indicated treatment times for qRT-PCR analysis of ACSL4 and GAPDH. After normalization with GAPDH, ACSL4 mRNA levels were plotted against the treatment time. D: Reporter constructs were cotransfected with a Renilla expression vector (pRL-SV40) into HepG2 cells. Two days post transfection, cell lysates were isolated to measure dual Luc activities. After normalization, the Luc activity of pGL3-basic is expressed as 1 and the Luc activity of pGL3-ACSL4 is expressed as fold of pGL3-basic. E: HepG2 cells were transfected with pGL3-ACSL4 and pRL-SV40 for 2 days prior to AA treatment of 24 h at the indicated doses. Cell lysates were isolated to measure dual Luc activities. The normalized Luc activity in control cells is expressed as 1. F: HepG2 cells were transfected with pcDNA-Luc control vector or pcDNA-Luc-ACSL4 3′UTR plasmid (ACSL4-UTR) for 1 day prior to AA treatment of 24 h at the indicated doses. Cell lysates were isolated to measure dual Luc activities. The normalized Luc activity of pCDNA-Luc in control cells is expressed as 1. n.s., not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109760&req=5

fig4: AA reduces ACSL4 protein half-life without affecting ACSL4 gene transcription or mRNA stability. A: HepG2 cells were treated with 50 μM AA or vehicle for 8 h. CHX, at 5 μg/ml concentration, was added to cells for the indicated times. Total cell lysates were subjected to Western blotting and bands were visualized with antibody against ACSL4 or β-actin. The indicated value is for the blot shown. B: After normalization to β-actin, the ACSL4 signal intensity was plotted against the CHX treatment time to calculate T1/2 of ACSL4 protein. The T1/2 data presented are the mean ± SEM of three independent CHX treatment experiments. C: HepG2 cells were treated with 50 μM AA or vehicle for 8 h. Then, actinomycin D (Act D), at a concentration of 5 μg/ml, was added to the cells and total RNA was isolated at the indicated treatment times for qRT-PCR analysis of ACSL4 and GAPDH. After normalization with GAPDH, ACSL4 mRNA levels were plotted against the treatment time. D: Reporter constructs were cotransfected with a Renilla expression vector (pRL-SV40) into HepG2 cells. Two days post transfection, cell lysates were isolated to measure dual Luc activities. After normalization, the Luc activity of pGL3-basic is expressed as 1 and the Luc activity of pGL3-ACSL4 is expressed as fold of pGL3-basic. E: HepG2 cells were transfected with pGL3-ACSL4 and pRL-SV40 for 2 days prior to AA treatment of 24 h at the indicated doses. Cell lysates were isolated to measure dual Luc activities. The normalized Luc activity in control cells is expressed as 1. F: HepG2 cells were transfected with pcDNA-Luc control vector or pcDNA-Luc-ACSL4 3′UTR plasmid (ACSL4-UTR) for 1 day prior to AA treatment of 24 h at the indicated doses. Cell lysates were isolated to measure dual Luc activities. The normalized Luc activity of pCDNA-Luc in control cells is expressed as 1. n.s., not significant.
Mentions: To determine whether AA lowering of ACSL4 protein levels was due to its increased degradation, we treated HepG2 cells with or without AA and/or with or without a protein synthesis inhibitor, CHX, and changes in ACSL4 protein levels were followed for the next 8 h by the Western blotting. Three separate experiments with identical conditions were performed. Figure 4A is a representative Western blot analysis. Quantitative data presented in Fig. 4B are derived from three independent experiments. AA treatment caused an accelerated degradation of ACSL4 protein with T1/2 = 4.2 ± 0.35 h as compared with T1/2 = 17.3 ± 1.84 h (P < 0.01) calculated from non-AA-treated control cells.

Bottom Line: AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis.We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination.Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304.

ABSTRACT
ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. Although an association between elevated levels of ACSL4 and hepatosteatosis has been reported, the function of ACSL4 in hepatic FA metabolism and the regulation of its functional expression in the liver remain poorly defined. Here we provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis. The inhibitory action of AA on ACSL4 protein stability could not be prevented by rosiglitazone or inhibitors that interfere with the cellular pathways involved in AA metabolism to biologically active compounds. In contrast, treatment of cells with inhibitors specific for the proteasomal degradation pathway largely prevented the AA-induced ACSL4 degradation. We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination. Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

No MeSH data available.


Related in: MedlinePlus