Participation of ATP7A in macrophage mediated oxidation of LDL

Abstract

ATP7A primarily functions to egress copper from cells, thereby supplying this cofactor to secreted copper-accepting enzymes. This ATPase has attracted significant attention since the discovery of its mutation leading to human Menkes disease and the demonstration of its distribution in various tissues. Recently, we reported that ATP7A is expressed in the human vasculature. In the present study, we investigated the cellular expression of ATP7A in atherosclerotic lesions of LDL receptor (-/-) mice. Subsequently, we examined the role of ATP7A in regulating the oxidation of LDL in a macrophage cell model. We observed that ATP7A is expressed in atherosclerotic murine aorta and colocalizes with macrophages. To investigate the function of ATP7A, we downregulated ATP7A expression in THP-1 derived macrophages using small interfering RNA (siRNA). ATP7A downregulation attenuated cell-mediated oxidation of LDL. Moreover, downregulation of ATP7A resulted in decreased expression and enzymatic activity of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), a key intracellular enzyme involved in cell-mediated LDL oxidation. In addition, cPLA(2)alpha promoter activity was decreased after downregulation of ATP7A, suggesting that ATP7A transcriptionally regulates cPLA(2)alpha expression. Finally, cPLA(2)alpha overexpression increased LDL oxidation, which was blocked by coadministration of ATP7A siRNA oligonucleotides. These findings suggest a novel mechanism linking ATP7A to cPLA(2)alpha and LDL oxidation, suggesting that this copper transporter could play a previously unrecognized role in the pathogenesis of atherosclerosis.

Fig. 1.

ATP7A is expressed in macrophages of aortic intimal lesions from LDLR^−/− mice. Aortic sections from LDLR ^−/−mice maintained on a Western-type high-fat/-cholesterol diet for 12 weeks were immunostained with antibodies against ATP7A and MOMA as described in Materials and Methods. Overlay of ATP7A (red) and MOMA (green) signals shows colocalization (yellow signal) in aortic intimal lesions. Images are representative of three separate experiments.

Fig. 2.

A: Effect of siRNA on ATP7A protein expression in human THP-1-derived macrophages. THP-1-derived macrophages (5 × 10⁵ cells/ml) were incubated with either 5 nM of ATP7A-specific (lanes 5 and 6) or control (lanes 3 and 4) siRNA oligonucleotides for 24 h. Control cells (lanes 1 and 2) were exposed to transfectant without siRNA oligonucleotides. The protein level of ATP7A was determined by Western blot, with α tubulin used as a loading control. Representative blots (upper panel) are from five individual experiments, and quantification is shown in the lower panel. *P < 0.01 versus ATP7A-downregulated macrophages. B: Effect of ATP7A downregulation on cell mediated LDL oxidation. After incubation with ATP7A or control siRNA oligonucleotides for 24 h, THP-1-derived macrophages were incubated with native LDL (250 µg/ml protein) in Ham's F12 medium without FBS for the indicated time periods. Following incubation, lipid oxidation was assessed by the TBA assay as described in the Materials and Methods. Each data point is the mean of triplicate measurements after subtraction of cell-free control. * P < 0.05 versus ATP7A-downregulated macrophages. ** P < 0.01 versus ATP7A-downregulated macrophages.

Fig. 3.

Comparison of LDL oxidation mediated by cells versus conditioned medium in THP-1-derived macrophages. After incubation with ATP7A or control siRNA oligonucleotides for 48 h, the conditioned media (left two bars) from THP-1-derived macrophages were harvested and then incubated with native LDL (250 µg/ml protein) in Ham's F12 medium without FBS for an additional 48 h. Afterwards, lipid oxidation was assessed by the TBA assay, while cell mediated LDL oxidation (right two bars) was performed as described in Fig. 2B. Each data point is the mean of triplicate measurements after subtraction of cell-free control. *P < 0.01 versus ATP7A-downregulated macrophages. ns, Not significant.

Fig. 4.

Effect of downregulation of ATP7A on mRNA levels (A), protein levels (B), enzymatic activity (C), and promoter activity (D) of cPLA₂α in human THP-1-derived macrophages. A: Following treatment with ATP7A-specific or control siRNA oligonucleotides for 24 h, total RNA was isolated, and human cPLA₂α and GAPDH expression was quantified by RT-PCR. The abundance of target mRNA was calculated in relation to the GAPDH mRNA in the same sample. The data are presented as fold change in mRNA expression compared with control cells (treated with control siRNA oligonucleotides). Each data point is the mean of triplicate measurements. *P < 0.05 versus ATP7A-downregulated macrophages. B and C: Following treatment with ATP7A-specific or control siRNA oligonucleotides for 48 h, cell lysates were prepared and the protein expression and activity of cPLA₂α was assessed as described in Fig. 2. For Western blot, α-tubulin served as a loading control. Representative blots are from three separate experiments. *P < 0.05 versus ATP7A-downregulated macrophages. D: Cells were cotransfected with the cPLA₂α-luciferase reporter plasmid (PA3-Luc/cPLA₂α) along with control or ATP7A siRNA oligonucleotides. Forty-eight hours later, lysates were prepared, and luciferase activity (normalized to phRLTK activity) was determined as described in Materials and Methods. Results represent the mean of three independent experiments. *P < 0.01 versus ATP7A-downregulated cells.

Fig. 5.

Effects of ATP7A downregulation on LDL oxidation in cells overexpressing cPLA₂α. Cells were cotransfected with the ECGF-cPLA₂α vector or control vector along with siRNA oligonucleotides targeted to ATP7A or control. Forty-eight hours later, cells were incubated with native LDL (250 µg/ml protein) in Ham's F12 medium without FBS for additional 48 h. Afterwards, lipid oxidation was assessed by the TBA assay. Each data point is the mean of triplicate measurements after subtraction of cell-free control. * P < 0.05 versus ATP7A-downregulated or control macrophages.