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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

Dose- and time-dependent downregulation of ACSL4 protein by AA. A, B: HepG2 cells were incubated for 24 h with the indicated concentrations of exogenous FA. ACSL4 protein levels were detected by Western blotting. After Western blotting, the protein amount of ACSL4 was quantified with the Alpha View software with normalization by signals of β-actin. The data in (A) are representative of three separate experiments. The indicated value is for the blot shown. The data presented in (B) are the mean ± SEM of three independent treatment experiments. C, D: Mouse primary hepatocytes were treated with 150 μM of each FA for 24 h. ACSL4 protein levels were detected by Western blotting. The data presented in (D) are the mean ± SEM of three independent treatment experiments. E, F: ACSL4 protein levels were examined in HepG2 cells that were treated with 150 μM AA for the indicated times. The Western blotting result shown in (E) is representative of three separate assays, and the quantitative results are presented as the mean ± SEM of three independent kinetic studies. C, control.
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fig3: Dose- and time-dependent downregulation of ACSL4 protein by AA. A, B: HepG2 cells were incubated for 24 h with the indicated concentrations of exogenous FA. ACSL4 protein levels were detected by Western blotting. After Western blotting, the protein amount of ACSL4 was quantified with the Alpha View software with normalization by signals of β-actin. The data in (A) are representative of three separate experiments. The indicated value is for the blot shown. The data presented in (B) are the mean ± SEM of three independent treatment experiments. C, D: Mouse primary hepatocytes were treated with 150 μM of each FA for 24 h. ACSL4 protein levels were detected by Western blotting. The data presented in (D) are the mean ± SEM of three independent treatment experiments. E, F: ACSL4 protein levels were examined in HepG2 cells that were treated with 150 μM AA for the indicated times. The Western blotting result shown in (E) is representative of three separate assays, and the quantitative results are presented as the mean ± SEM of three independent kinetic studies. C, control.

Mentions: To identify specific FA species that affect ACSL4 protein expression, we treated HepG2 cells with the indicated concentrations of individual FAs of varying chain length and degree of saturation (19). PA or OA exposure of cells did not produce a suppressive effect on ACSL4 protein levels, whereas inclusion of AA in the culture medium greatly suppressed the ACSL4 protein abundance in a dose-dependent manner. Figure 3A is a representative Western blot analysis. Quantitative data presented in Fig. 3B are derived from three independent experiments. These data show that a 5 μM concentration of AA caused lowering of ACSL4 protein levels by ∼30%, while a maximum reduction in ACSL4 protein levels was achieved at an AA concentration of 50 μM. None of the doses of AA applied had any significant effect on cell viability (supplementary Fig. IIA). Furthermore, ACSL4 mRNA levels were not reduced over the AA concentration range (supplementary Fig. IIB). In contrast, SREBP1c mRNA levels were dose-dependently lowered by AA treatment in HepG2 cells, which was in line with literature reports (20). We also measured acyl-CoA synthesis activity and showed that treatment of HepG2 cells with AA (150 μM) for 24 h reduced the arachidonoyl-CoA synthetase activity by 46% (P < 0.01) (supplementary Fig. IIC). The specific inhibitory effect of AA on ACSL4 protein expression was also detected in mouse primary hepatocytes (Fig. 3C, D).


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)

Dose- and time-dependent downregulation of ACSL4 protein by AA. A, B: HepG2 cells were incubated for 24 h with the indicated concentrations of exogenous FA. ACSL4 protein levels were detected by Western blotting. After Western blotting, the protein amount of ACSL4 was quantified with the Alpha View software with normalization by signals of β-actin. The data in (A) are representative of three separate experiments. The indicated value is for the blot shown. The data presented in (B) are the mean ± SEM of three independent treatment experiments. C, D: Mouse primary hepatocytes were treated with 150 μM of each FA for 24 h. ACSL4 protein levels were detected by Western blotting. The data presented in (D) are the mean ± SEM of three independent treatment experiments. E, F: ACSL4 protein levels were examined in HepG2 cells that were treated with 150 μM AA for the indicated times. The Western blotting result shown in (E) is representative of three separate assays, and the quantitative results are presented as the mean ± SEM of three independent kinetic studies. C, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Dose- and time-dependent downregulation of ACSL4 protein by AA. A, B: HepG2 cells were incubated for 24 h with the indicated concentrations of exogenous FA. ACSL4 protein levels were detected by Western blotting. After Western blotting, the protein amount of ACSL4 was quantified with the Alpha View software with normalization by signals of β-actin. The data in (A) are representative of three separate experiments. The indicated value is for the blot shown. The data presented in (B) are the mean ± SEM of three independent treatment experiments. C, D: Mouse primary hepatocytes were treated with 150 μM of each FA for 24 h. ACSL4 protein levels were detected by Western blotting. The data presented in (D) are the mean ± SEM of three independent treatment experiments. E, F: ACSL4 protein levels were examined in HepG2 cells that were treated with 150 μM AA for the indicated times. The Western blotting result shown in (E) is representative of three separate assays, and the quantitative results are presented as the mean ± SEM of three independent kinetic studies. C, control.
Mentions: To identify specific FA species that affect ACSL4 protein expression, we treated HepG2 cells with the indicated concentrations of individual FAs of varying chain length and degree of saturation (19). PA or OA exposure of cells did not produce a suppressive effect on ACSL4 protein levels, whereas inclusion of AA in the culture medium greatly suppressed the ACSL4 protein abundance in a dose-dependent manner. Figure 3A is a representative Western blot analysis. Quantitative data presented in Fig. 3B are derived from three independent experiments. These data show that a 5 μM concentration of AA caused lowering of ACSL4 protein levels by ∼30%, while a maximum reduction in ACSL4 protein levels was achieved at an AA concentration of 50 μM. None of the doses of AA applied had any significant effect on cell viability (supplementary Fig. IIA). Furthermore, ACSL4 mRNA levels were not reduced over the AA concentration range (supplementary Fig. IIB). In contrast, SREBP1c mRNA levels were dose-dependently lowered by AA treatment in HepG2 cells, which was in line with literature reports (20). We also measured acyl-CoA synthesis activity and showed that treatment of HepG2 cells with AA (150 μM) for 24 h reduced the arachidonoyl-CoA synthetase activity by 46% (P < 0.01) (supplementary Fig. IIC). The specific inhibitory effect of AA on ACSL4 protein expression was also detected in mouse primary hepatocytes (Fig. 3C, D).

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