Korean J Physiol Pharmacol.  2015 Nov;19(6):507-514. 10.4196/kjpp.2015.19.6.507.

Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation

• Yang JY
• Park MY
• Park SY
• Yoo HI
• Kim MS
• Kim JH
• Kim WJ
• Jung JY

• KMID: 2070790
• DOI: http://doi.org/10.4196/kjpp.2015.19.6.507

Abstract

Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells.

Keyword

AMP-activated protein kinase; Autophagy; Cytoprotection; MC3T3-E1 cells; Nitric oxide

MeSH Terms

Acridine Orange
AMP-Activated Protein Kinases*
Apoptosis
Autophagy*
Blotting, Western
Bone Remodeling
Caspase 3
Cell Death
Cell Survival
Cytoprotection*
Humans
In Situ Nick-End Labeling
Nitric Oxide
Nitroprusside
Osteoblasts
Phosphotransferases
Tissue Donors
Vacuoles
AMP-Activated Protein Kinases
Acridine Orange
Caspase 3
Nitric Oxide
Nitroprusside
Phosphotransferases

Figure

• 

  Fig. 1 NO decreases cell viability in MC3T3-E1 cells. Cell viability was determined by MTT assay. The MC-3T3-E1 cells were incubated with different concentrations of SNP for 24 hrs (A) and 1.5 mM SNP for indicated times (B). Each value represents the mean of three replicates. *p<0.05, **p<0.01 and ***p<0.001 vs. the control.

• 

  Fig. 2 Effect of NO on the expression of autophagy markers in MC3T3-E1 cells. Cells were incubated with 1.5 mM SNP for the indicated times and analyzed by western blot analysis using antibodies for the indicated proteins (A). β-actin was used as the loading control. The MC3T3-E1 cells plated in glass chamber slides were either pretreated without or with 3MA, an autophagy inhibitor, before being treated with SNP. The cells were then fixed and stained with acridine orange and analyzed by fluorescence microscopy (B). The formation of autophagosome was shown in puncta fluorescence (red). Scale bars, 100 µm.

• 

  Fig. 3 Cytoprotective role of NO-induced autophagy in MC3T3-E1 cells. The cells were cultured with 1.5 mM SNP without or with 5 mM 3MA for 24 hrs. Cell viability was detected by MTT assay (A). In cells co-treated with SNP and 3MA, the cell viability was lower than that in MC3T3-E1 cells treated with SNP alone. p62, LC3, ATG7, cleaved caspase-3 and PARP levels were determined using western blot analysis after MC3T3-E1 cells were exposed to 1.5 mM SNP with (+) or without (-) pretreatment with 5 mM 3MA for 1 hr (B). β-actin was used as loading control. Each value represents the mean of three replicates. ***p<0.001 vs. the untreated control, #p<0.05 vs. the SNP-treated group.

• 

  Fig. 4 Effect of NO on AMPK signaling in MC3T3-E1 cells. The phosphorylation of AMPK in 1.5 mM SNP-treated MC3T3-E1 cells during 12 hrs was determined by western blot analysis. β-actin was used as the loading control. Values are expressed as mean±SD of three separate experiments. **p<0.01, ***p<0.001 vs. the untreated control.

• 

  Fig. 5 Cytoprotective role of NO-activated AMPK in MC3T3-E1 cells. The cell were cultured with 1.5 mM SNP without or with 10 µM compound C, an AMPK inhibitor, for 24 hrs. Cell viability was detected by MTT assay (A) and cell death was evaluated by trypan blue staining (B). Values are expressed as mean±SD of three replicates. ***p<0.001, **p<0.01 vs. the untreated control, #p<0.05, ##p<0.01 vs. the S NP-treated group. (C) Cleaved protein levels of caspase-3 and PARP were determined using western blot analysis after the MC3T3-E1 cells were exposed to 1.5 mM of SNP with or without pretreatment with 10 µM of compound C. (D) Cells plated in glass chamber slides were either pretreated with (+) or without (-) compound C prior to SNP treatment. The cells were then fixed and labeled with TUNEL and analyzed by fluorescence microscopy. TUNEL positive cells in the fluorescence (green) were increased by compound C. Arrows indicate the TUNEL positive cells. Scale bars, 50 µm.

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