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. 2014 Oct;131(1):21-32.
doi: 10.1111/jnc.12780. Epub 2014 Jul 10.

Induction of serpinb1a by PACAP or NGF Is Required for PC12 Cells Survival After Serum Withdrawal

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Free PMC article

Induction of serpinb1a by PACAP or NGF Is Required for PC12 Cells Survival After Serum Withdrawal

Tommy Seaborn et al. J Neurochem. .
Free PMC article

Abstract

PC12 cells are used to study the signaling mechanisms underlying the neurotrophic and neuroprotective activities of pituitary adenylate cyclase-activating polypeptide (PACAP) and nerve growth factor (NGF). Previous microarray experiments indicated that serpinb1a was the most induced gene after 6 h of treatment with PACAP or NGF. This study confirmed that serpinb1a is strongly activated by PACAP and NGF in a time-dependent manner with a maximum induction (~ 50-fold over control) observed after 6 h of treatment. Co-incubation with PACAP and NGF resulted in a synergistic up-regulation of serpinb1a expression (200-fold over control), suggesting that PACAP and NGF act through complementary mechanisms. Consistently, PACAP-induced serpinb1a expression was not blocked by TrkA receptor inhibition. Nevertheless, the stimulation of serpinb1a expression by PACAP and NGF was significantly reduced in the presence of extracellular signal-regulated kinase, calcineurin, protein kinase A, p38, and PI3K inhibitors, indicating that the two trophic factors share some common pathways in the regulation of serpinb1a. Finally, functional investigations conducted with siRNA revealed that serpinb1a is not involved in the effects of PACAP and NGF on PC12 cell neuritogenesis, proliferation or body cell volume but mediates their ability to block caspases 3/7 activity and to promote PC12 cell survival.

Keywords: caspase; cell death; cell survival; nerve growth factor; neuroprotection; pituitary adenylate cyclase-activating polypeptide.

Conflict of interest statement

Authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Effects of PACAP and NGF treatment on serpinb1a mRNA expression in PC12 cells. (A) Time-course effect: Cells were cultured in the presence of PACAP (10−7 M) or NGF (100 ng/ml) for durations ranging from 0 to 48 hours. (B) Dose effect: Cells were cultured 6 hours in the presence of PACAP (10−12 to 10−7 M) or NGF (10−12 to 10−7 g/ml). (C) Combined effect: Cells were cultured 6 hours in the presence of PACAP (10−7 M) and/or NGF (100 ng/ml). Quantitative PCR results were corrected using the GAPDH signal as internal control and expressed as the mean fold induction (± S.E.M.) of serpinb1a mRNA level compared to the control. * p<0.05 and ** p<0.01 versus control.
Fig. 2
Fig. 2
Effects of cAMP and PKC activation as well as VIP treatment on serpinb1a mRNA expression in PC12 cells. Cells were cultured 6 h in the presence of PACAP (10−7 M), the cAMP activator forskolin (25 µM), the cAMP analog dbcAMP (10−3 M), the PKC activator PMA (10−7 M), a combination of PMA plus forskolin, or VIP (10−7 M). Quantitative PCR results were corrected using the GAPDH signal as internal control and expressed as the mean fold induction (± S.E.M.) of serpinb1a mRNA level compared to the control without activator. * p<0.05 and ** p<0.01 versus control.
Fig. 3
Fig. 3
Investigation of the transduction pathways involved in the regulation of serpinb1a expression by PACAP and NGF in PC12 cells. Cells were pre-incubated for 30 min with inhibitors and then cultured for 6 h in the presence of PACAP (10−7 M) or NGF (100 ng/ml). (A) Effect of protein kinase inhibitors on PACAP- and NGF-induced serpinb1a expression. Cells were pre-incubated with the PKA inhibitor H89 (10 µM), the PKC inhibitor Gö6983 (1 mM), or the PKA/PKC inhibitor H7 (50 µM). (B) Effect of TrkA and PI3K inhibitors on PACAP- and NGF-induced serpinb1a expression. Cells were pre-incubated with the TrkA inhibitor K252a (1 µM) or the PI3K inhibitor LY294002 (20 µM). (C) Effect of MAP kinase inhibitors on PACAP- and NGF-induced serpinb1a expression. Cells were pre-incubated with the MEK inhibitors PD98059 (50 µM) or U0126 (25 µM), the p38 inhibitor SB203580 (10 µM), or the JNK inhibitor I (2 µM). (D) Effect of calcineurin inhibitors and calcium channel blockers on PACAP- and NGF-induced serpinb1a expression. Cells were pre-incubated with the calcineurin inhibitors ascomycin (100 nM) or cyclosporin A (100 nM), the FKBP12 inhibitor rapamycin (100 nM), or the calcium channel blocker D600 (30 µM). Quantitative PCR results were corrected using the GAPDH signal as internal control and expressed as the mean fold induction (± S.E.M.) of serpinb1a mRNA level compared to the control without inhibitor. ** p<0.01 versus control; # p<0.05 and ## p<0.01 versus PACAP without inhibitor; § p<0.05 and §§ p<0.01 versus NGF without inhibitor; NS, not significantly different from corresponding treatment without inhibitor.
Fig. 4
Fig. 4
Involvement of serpinb1a in the neurotrophic effects of PACAP and NGF on PC12 cells. Cells were transfected with control siRNA or siRNA directed against serpinb1a mRNA, and were treated with PACAP (10−7 M) or NGF (100 ng/ml) 2 days later. (A) Effect of anti-serpinb1a siRNA on cell differentiation (photomicrographs) and serpinb1a mRNA expression after treatment with PACAP or NGF for 24 h and 6 h, respectively. Quantitative PCR results were corrected using the GAPDH signal as internal control and expressed as the mean fold induction (± S.E.M.) of serpinb1a mRNA level compared to untreated cells transfected with control siRNA. (B) Neuritogenesis assessment by measuring the percentage of cells with neurites, the number of neurites per cell, and the overall neurite outgrowth after 48 h of treatment with PACAP or NGF in the presence of control siRNA or anti-serpinb1a siRNA. (C) Quantification of cell proliferation and cell size by counting the number of cells and the percentage of cells with a diameter greater than 17 µm after 48 hours of treatment with PACAP or NGF in the presence of control siRNA or anti-serpinb1a siRNA. * p<0.05 and ** p<0.01 versus control in the presence of control siRNA; ## p<0.01 versus PACAP in the presence of control siRNA; §§ p<0.01 versus NGF in the presence of control siRNA. Scale bar = 15 µm.
Fig. 5
Fig. 5
Involvement of serpinb1a in PACAP- and NGF-induced survival of PC12 cells. Cells were transfected with control siRNA or siRNA directed against serpinb1a mRNA and were serum-deprived 2 days later. After 12 h in serum-free conditions, cells were treated with PACAP (10−7 M) or NGF (100 ng/ml). (A) Quantification of caspases-3/7 activity after 6 h of treatment with PACAP or NGF. (B) Typical microphotographs illustrating the effect of anti-serpinb1a siRNA on cell survival. Two days after PACAP or NGF treatment, living cells were labeled with calcein (green fluorescence) and dead cells were labeled with ethidium homodimer-1 (red fluorescence). (C) Quantification of cell survival by counting the number of cells after 48 h of treatment with PACAP or NGF in serum-deprived conditions. * p<0.05 versus control in the presence of control siRNA; # p<0.05 versus PACAP in the presence of control siRNA; § p<0.05 versus NGF in the presence of control siRNA. Scale bar = 100 µm.

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