Endocrinol Metab.  2016 Jun;31(2):336-342. 10.3803/EnM.2016.31.2.336.

The Role of Nuclear Factor-E2-Related Factor 1 in the Oxidative Stress Response in MC3T3-E1 Osteoblastic Cells

• Park SY
• Kim SH
• Yoon HK
• Yim CH
• Lim SK

• KMID: 2308865
• DOI: http://doi.org/10.3803/EnM.2016.31.2.336

Abstract

BACKGROUND
Reactive oxygen species (ROS) and antioxidants are associated with maintenance of cellular function and metabolism. Nuclear factor-E2-related factor 1 (NFE2L1, Nrf1) is known to regulate the expression of a number of genes involved in oxidative stress and inflammation. The purpose of this study was to examine the effects of NFE2L1 on the response to oxidative stress in osteoblastic MC3T3-E1 cells.
METHODS
The murine calvaria-derived MC3T3-E1 cell line was exposed to lipopolysaccharide (LPS) for oxidative stress induction. NFE2L1 effects were evaluated using small interfering RNA (siRNA) for NFE2L1 mRNA. ROS generation and the levels of known antioxidant enzyme genes were assayed.
RESULTS
NFE2L1 expression was significantly increased 2.4-fold compared to the control group at 10 µg/mL LPS in MC3T3-E1 cells (P<0.05). LPS increased formation of intracellular ROS in MC3T3-E1 cells. NFE2L1 knockdown led to an additional increase of ROS (20%) in the group transfected with NFE2L1 siRNA compared with the control group under LPS stimulation (P<0.05). RNA interference of NFE2L1 suppressed the expression of antioxidant genes including metallothionein 2, glutamatecysteine ligase catalytic subunit, and glutathione peroxidase 1 in LPS-treated MC3T3-E1 cells.
CONCLUSION
Our results suggest that NFE2L1 may have a distinct role in the regulation of antioxidant enzymes under inflammation-induced oxidative stress in MC3T3-E1 osteoblastic cells.

Keyword

NF-E2-related factor 1; Oxidative stress; Osteoblasts

MeSH Terms

Antioxidants
Catalytic Domain
Cell Line
Glutathione Peroxidase
Inflammation
Metabolism
Metallothionein
NF-E2-Related Factor 1
Osteoblasts*
Oxidative Stress*
Reactive Oxygen Species
RNA Interference
RNA, Messenger
RNA, Small Interfering
Antioxidants
Glutathione Peroxidase
Metallothionein
NF-E2-Related Factor 1
RNA, Messenger
RNA, Small Interfering
Reactive Oxygen Species

Figure

• 

  Fig. 1 The effect of lipopolysaccharide (LPS) on nuclear factor-E2-related factor 1 (NFE2L1) mRNA expression in MC3T3-E1 cells. Cells were treated with 0, 2, 5, or 10 µg/mL LPS for 24 hours. Levels of mRNA were analyzed by (A) semi-quantitative polymerase chain reaction (PCR) and (B) quantitative real time-PCR. The expression level of each mRNA was normalized to the β-actin levels. aP<0.05 compared with the control group.

• 

  Fig. 2 Nuclear factor-E2-related factor 1 (NFE2L1) mRNA expression after transient transfection with control siRNA (siCONT) or NFE2L1 siRNA (siNFE2L1) in MC3T3-E1 cells. Levels of mRNA were analyzed by semi-quantitative (A) polymerase chain reaction (PCR) and (B) quantitative real time-PCR. The expression level of each mRNA was normalized to the β-actin levels. aP<0.05 compared with the siCONT group.

• 

  Fig. 3 Measurement of reactive oxygen species (ROS) with H2DCF-DA in MC3T3-E1 cells. Intracellular ROS in the transfectants of control siRNA (siCONT) and nuclear factor-E2-related factor 1 siRNA (siNFE2L1) were compared under control (no stimulation) and stimulations by lipopolysaccharide (LPS, 10 µg/mL) for 10 minutes. aP<0.05 compared with the control group or siCONT cells.

• 

  Fig. 4 The effect of nuclear factor-E2-related factor 1 (NFE2L1) knockdown on antioxidant gene mRNA expression in lipopolysaccharide (LPS) treated cells. Antioxidant genes are as follows: (A) metallothionein 1 (MT1), (B) metallothionein 2 (MT2), (C) glutamate-cysteine ligase catalytic subunit (GCLC), (D) glutamate-cysteine ligase modifier subunit (GCLM), (E) NAD(P)H dehydrogenase, quinone 1 (NQO1), and (F) glutathione peroxidase 1 (GPx1). MC3T3-E1 cells were transfected with control siRNA (siCONT) or NFE2L1 siRNA (siNFE2L1) followed by 24-hour treatment of 10 µg/mL LPS. Controls received culture medium only. Quantitation of mRNA levels was analyzed by quantitative real-time polymerase chain reaction. The expression level of each mRNA was normalized to the β-actin levels. aP<0.05 compared with the control group or siCONT cells.

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