Immune Netw.  2014 Apr;14(2):116-122. 10.4110/in.2014.14.2.116.

The Nuclear Orphan Receptor NR4A1 is Involved in the Apoptotic Pathway Induced by LPS and Simvastatin in RAW 264.7 Macrophages

• Kim YC
• Song SB
• Lee SK
• Park SM
• Kim YS

• KMID: 2168022
• DOI: http://doi.org/10.4110/in.2014.14.2.116

Abstract

Macrophage death plays a role in several physiological and inflammatory pathologies such as sepsis and arthritis. In our previous work, we showed that simvastatin triggers cell death in LPS-activated RAW 264.7 mouse macrophage cells through both caspase-dependent and independent apoptotic pathways. Here, we show that the nuclear orphan receptor NR4A1 is involved in a caspase-independent apoptotic process induced by LPS and simvastatin. Simvastatin-induced NR4A1 expression in RAW 264.7 macrophages and ectopic expression of a dominant-negative mutant form of NR4A1 effectively suppressed both DNA fragmentation and the disruption of mitochondrial membrane potential (MMP) during LPS- and simvastatin-induced apoptosis. Furthermore, apoptosis was accompanied by Bcl-2-associated X protein (Bax) translocation to the mitochondria. Our findings suggest that NR4A1 expression and mitochondrial translocation of Bax are related to simvastatin-induced apoptosis in LPS-activated RAW 264.7 macrophages.

Keyword

NR4A1; Apoptosis; LPS; Simvastatin; Macrophages

MeSH Terms

Animals
Apoptosis
Arthritis
bcl-2-Associated X Protein
Cell Death
Child
Child, Orphaned*
DNA Fragmentation
Humans
Macrophages*
Membrane Potential, Mitochondrial
Mice
Mitochondria
Pathology
Sepsis
Simvastatin*
Simvastatin
bcl-2-Associated X Protein

Figure

• 

  Figure 1 Induction of apoptosis by LPS and simvastatin. (A) Raw264.7 cells were treated with LPS and simvastatin with FTI-276, GGTI-286, Y-27632 for 14 h, and subgenomic contents were analyzed by PI staining and flow cytometry. (B) Mevalonate pathway and inhibitors used in this study. The enzymatic target steps of simvastatin (HMG-CoA reductase inhibitor), FTI-276 (farnesyl transferase inhibitor), GGTI-286 (geranylgeranyl transferase inhibitor), and Y-27632 (Rho kinase inhibitor) were displayed.

• 

  Figure 2 Induction of NR4A1 mRNA by LPS and simvastatin. (A) RAW 264.7 cells were treated with LPS and/or simvastatin alone or in combination with mevalonate or benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)-fluoromethylketone (Z-VAD-FMK) for 12 h and the level of nuclear orphan receptor NR4A1 mRNA was measured using RT-PCR. Total RNA was extracted and amplified as described in the Materials and Methods. GAPDH was used to normalize the quantity of amplified NR4A1. (B) For the kinetic analysis of NR4A1 expression, cells were treated with 1µg/ml LPS or 2µM simvastatin in combination with 20µM Z-VAD-FMK for indicated times (up to 12 h) and were analyzed using RT-PCR.

• 

  Figure 3 The effect of dominant-negative NR4A1 expression on apoptosis. (A) RAW 264.7 cells were transfected with control or dominant-negative NR4A1 (NR4A1-DN) vectors and treated with various concentrations of LPS and 2µM simvastatin for 12 h. Using flow cytometry, the percentage of subgenomic DNA content was measured in propidium iodide (PI)-stained cells to evaluate DNA fragmentation. The inset shows the presence of NR4A1-DN in the nucleus of transfected cells. (B) RAW 264.7 cells transfected with control or NR4A1-DN vectors were incubated in LPS or simvastatin alone or in combination with Z-VAD-FMK for 12 h and stained using rhodamine-123. The mitochondrial membrane potential (MMP) of the stained cells was analyzed using flow cytometry. A representative result of four experiments is shown.

• 

  Figure 4 Translocation of Bax to mitochondria during simvastatin-triggered apoptosis in LPS-activated cells. RAW 264.7 cells were incubated with LPS or simvastatin in combination with mevalonate or Z-VAD-FMK for 11 h. Cell extracts were separated into cytosolic and mitochondrial fractions. For SDS-PAGE separation, 4µg and 2µg of cytosolic and mitochondrial protein of cytosolic and mitochondrial protein, respectively, were loaded and Bax protein in each fraction was detected by western blot. Cy, cytosolic protein; Mi, mitochondrial protein.

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