Intermittent hypoxia-induced protein phosphatase 2A activation reduces PC12 cell proliferation and differentiation

Abstract

Background: Intermittent hypoxia (IH) plays a critical role in sleep breathing disorder-associated hippocampus impairments, including neurocognitive deficits, irreversible memory and learning impairments. IH-induced neuronal injury in the hippocampus may result from reduced precursor cell proliferation and the relative numbers of postmitotic differentiated neurons. However, the mechanisms underlying IH-induced reactive oxygen species (ROS) generation effects on cell proliferation and neuronal differentiation remain largely unknown.

Results: ROS generation significantly increased after 1-4 days of IH without increased pheochromocytoma-12 (PC12) cell death, which resulted in increased protein phosphatase 2A (PP2A) mRNA and protein levels. After 3-4 days of IH, extracellular signal-regulated kinases 1/2 (ERK1/2) protein phosphorylation decreased, which could be reversed by superoxide dismutase (SOD), 1,10-phenanthroline (Phe), the PP2A phosphorylation inhibitors, okadaic acid (OKA) and cantharidin, and the ERK phosphorylation activator nicotine (p < 0.05). In particular, the significantly reduced cell proliferation and increased proportions of cells in the G0/G1 phase after 1-4 days of IH (p < 0.05), which resulted in decreased numbers of PC12 cells, could be reversed by treatment with SOD, Phe, PP2A inhibitors and an ERK activator. In addition, the numbers of nerve growth factor (NGF)-induced PC12 cells with neurite outgrowths after 3-4 days of IH were less than those after 4 days of RA, which was also reversed by SOD, Phe, PP2A inhibitors and an ERK activator.

Conclusions: Our results suggest that IH-induced ROS generation increases PP2A activation and subsequently downregulates ERK1/2 activation, which results in inhibition of PC12 cell proliferation through G0/G1 phase arrest and NGF-induced neuronal differentiation.

Figure 1

Intermittent hypoxia (IH) effects on mitochondrial reactive oxygen species (ROS) generation and cell death in PC12 cells. (A) Mitochondrial ROS generation was determined by flow cytometry using MitoSOX Red. (B) Quantitative levels of mitochondrial ROS generation in PC12 cells exposed to normoxia for 4 days (RA4, n = 16), IH for 1–4 days (IH1, n = 6; IH2, n = 6; IH3, n = 7; IH4, n = 7) and IH4 along with the superoxide dismutase (IH4 + SOD, n = 6) and Mn(III)tetrakis(4-benzoic acid)porphyrin (IH4 + MnTBAP, n = 6). (C-E) Percentages of viable cells (M1), apoptotic fractions (M2) and necrotic fractions (M3) were assessed using an Annexin V assay and flow cytomery. (F) Quantitative levels of viable, apoptotic and necrotic fractions among PC12 cells exposed to RA4 (n = 10), IH4 (n = 10) and RA4 along with H₂O₂ (RA4 + H₂O₂, n = 4). *p < 0.05 compared with RA4. ^#p < 0.05 compared with IH4. Values are means ± SEMs.

Figure 2

Intermittent hypoxia (IH) effects on protein phosphatase 2A (PP2A) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. (A) PP2A mRNA levels were determined by real-time quantitative polymerase chain reaction (qPCR) and (B) PP2A protein levels were determined by Western blot in PC12 cells after 4 days of room air exposure (RA4, n = 7), 3–4 days of IH (IH3, n = 5; IH4, n = 5), and IH4 along with superoxide dismutase (IH4 + SOD, n = 6), 1,10-phenanthroline (IH4 + Phe, n = 5) or okadaic acid (IH4 + OKA, n = 5). (C) ERK1/2 phosphorylation levels in PC12 cells were determined by Western blotting after RA4, IH3, IH4, IH4 + SOD, IH4 + Phe, IH4 + OKA and (D) immunocytofluorescence (green) after RA4, IH4, IH4 along with cantharidin (IH4 + Can, n = 5) and nicotine (IH4 + Nicotine, n = 5). *p < 0.05 compared with RA4. ^#p < 0.05 compared with IH4. Values are means ± SEMs.

Figure 3

Intermittent hypoxia (IH) effects on PC12 cell numbers, cell viability, cell proliferation and cell cycle progression. (A) Numbers of PC12 cells as evaluated using Hoechst staining (blue) and confocal microscopy. (B) Quantitative PC12 cell numbers after exposure to normoxia (RA) and IH for 1–4 days (n = 5 for per group). (C) PC12 cell numbers as determined by MTT assay after exposure to RA and IH for 1–4 days (n = 5 for per group). (D) PC12 cell proliferation determined by BrdU cell proliferation ELISA assay kit after exposure to 4 days of RA (RA4, n = 8) and IH (IH4, n = 8). (E) PC12 cell cycle progression after exposure to RA4 (n = 8), IH3 (n = 7) and IH4 (n = 7) as evaluated by propidium iodide staining and flow cytometry. Percentages of cells in G₀/G₁ phase arrest (E and F). *p < 0.05 compared with RA in (B) and (C) or RA4 in (D) and (F). Values are means ± SEMs.

Figure 4

Effect of intermittent hypoxia (IH)-induced ROS generation on PP2A activation and downregulated ERK1/2 activation leading to PC12 cell proliferation inhibition. (A) PC12 cell numbers determined by MTT assay after exposure to normoxia for 4 days (RA4, control, n = 16), and RA4 along with the ERK1/2 phosphorylation inhibitors U0126 (n = 8) and PD98059 (n = 7), the ERK1/2 phosphorylation activator nicotine (n = 14), and exposed to IH for 4 days (IH4, control, n = 8), IH4 along with superoxide dismutase (SOD, n = 12), the PP2A activation inhibitors 1,10-phenanthroline (Phe, n = 12), okadaic acid (OKA, n = 9) and cantharidin (Can, n = 9) and nicotine (n = 9). (B) PC12 cell proliferation determined by BrdU cell proliferation ELISA assay kit after exposure to RA4 (n = 8), IH4 (control, n = 8) and IH4 along with SOD (n = 7), Phe (n = 7), OKA (n = 7), Can (n = 7) and nicotine (n = 6). (C) Percentages of PC12 cells in G₀/G₁ phase arrest as determined by propidium iodide staining and flow cytometry after exposure to RA4 (control, n = 8), RA4 along with U0126 (n = 4), RA4 with nicotine (n = 4), IH4 (control, n = 7), IH4 along with SOD (n = 6), Phe (n = 5), OKA (n = 5) and nicotine (n = 4). *p < 0.05 compared with RA4. ^#p < 0.05 compared with IH4. Values are means ± SEMs.

Figure 5

Effects of intermittent hypoxia (IH)-induced ROS generation on PP2A activation and downregulated ERK1/2 activation leading cell differentiation inhibition by nerve growth factor (NGF)-stimulated PC12 cells. (A) Images of neurite outgrowth using light microscopy and (B) percentages of differentiated cells among NGF-stimulated PC12 cells after 3–4 days of intermittent hypoxia (n = 5 for per group). (C) Percentages of differentiated cells among NGF-stimulated PC12 cells after exposure to normoxia for 4 days (RA4, control, n = 16), RA4 along with U0126 (n = 6) and PD98059 (n = 5) (ERK1/2 phosphorylation inhibitors), and nicotine (n = 10) (ERK1/2 phosphorylation inducer), and exposed to IH for 4 days (IH4, control, n = 14), IH4 along with superoxide dismutase (SOD, n = 8), 1,10-phenanthroline (Phe, n = 9), okadaic acid (OKA, n = 12) and cantharidin (Can, n = 5) (PP2A activation inhibitors), and nicotine (n = 7). *p < 0.05 compared with RA4. ^#p < 0.05 compared with IH4. Values are means ± SEMs.

Figure 6

Possible molecular mechanisms for intermittent hypoxia (IH)-induced activation of protein phosphatase 2A (PP2A) and inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation contribute to inhibiting PC12 cell proliferation through G ₀ /G ₁ phase arrest and nerve growth factor (NGF)-induced neuronal differentiation.