Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor,thereby modulating activation of MAP kinase,Akt, and neurite outgrowth in PC12 cells

In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk(A). cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.     

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

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.     

Separate Cyclic AMP Sensors for Neuritogenesis,Growth Arrest,and Survival of Neuroendocrine Cells

Dividing neuroendocrine cells differentiate into a neuronal-like phenotype in response to ligands activating G protein-coupled receptors, leading to the elevation of the second messenger cAMP. Growth factors that act at receptor tyrosine kinases, such as nerve growth factor, also cause differentiation. We report here that two aspects of cAMP-induced differentiation, neurite extension and growth arrest, are dissociable at the level of the sensors conveying the cAMP signal in PC12 and NS-1 cells. Following cAMP elevation, neuritogenic cyclic AMP sensor/Rapgef2 is activated for signaling to ERK to mediate neuritogenesis, whereas Epac2 is activated for signaling to the MAP kinase p38 to mediate growth arrest. Neither action of cAMP requires transactivation of TrkA, the receptor for NGF. In fact, the differentiating effects of NGF do not require activation of any of the cAMP sensors protein kinase A, Epac, or neuritogenic cyclic AMP sensor/Rapgef2 but, rather, depend on ERK and p38 activation via completely independent signaling pathways. Hence, cAMP- and NGF-dependent signaling for differentiation are also completely insulated from each other. Cyclic AMP and NGF also protect NS-1 cells from serum withdrawal-induced cell death, again by two wholly separate signaling mechanisms, PKA-dependent for cAMP and PKA-independent for NGF.     

Nerve growth factor-mediated inhibition of apoptosis post-caspase activation is due to removal of active caspase-3 in a lysosome-dependent manner

Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner.     

Multiple G<Subscript>i</Subscript> Proteins Participate in Nerve Growth Factor-Induced Activation of c-Jun N-terminal Kinases in PC12 Cells

Nerve growth factor (NGF)-mediated activation of mitogen-activated protein kinases (MAPK) is critical for differentiation and apoptosis of PC12 cells. Since NGF employs stress-activated c-Jun N-terminal kinase (JNK) to regulate both programmed cell death and neurite outgrowth of PC12 cells, we examined NGF-regulated JNK activity and the role of G_i/o proteins. Induction of JNK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). To discern the participation of various signaling intermediates, PC12 cells were treated with specific inhibitors prior to NGF challenge. NGF-elevated JNK activity was abolished by inhibitors of JNK, p38 MAPK, Src, JAK3 and MEK1/2. NGF-dependent JNK phosphorylation became insensitive to PTX treatment upon transient expressions of Gα_z or the PTX-resistant mutants of Gα_i1–3 and Gα_oA. Collectively, these studies indicate that NGF-dependent JNK activity may be mediated via G_i1–3 proteins, JAK3, Src, p38 MAPK and the MEK/ERK cascade.     

CDK4-mediated MnSOD activation and mitochondrial homeostasis in radioadaptive protection

Mammalian cells are able to sense environmental oxidative and genotoxic conditions such as the environmental low-dose ionizing radiation (LDIR) present naturally on the earth’s surface. The stressed cells then can induce a so-called radioadaptive response with an enhanced cellular homeostasis and repair capacity against subsequent similar genotoxic conditions such as a high dose radiation. Manganese superoxide dismutase (MnSOD), a primary mitochondrial antioxidant in mammals, has long been known to play a crucial role in radioadaptive protection by detoxifying O₂^•− generated by mitochondrial oxidative phosphorylation. In contrast to the well-studied mechanisms of SOD2 gene regulation, the mechanisms underlying posttranslational regulation of MnSOD for radioprotection remain to be defined. Herein, we demonstrate that cyclin D1/cyclin-dependent kinase 4 (CDK4) serves as the messenger to deliver the stress signal to mitochondria to boost mitochondrial homeostasis in human skin keratinocytes under LDIR-adaptive radioprotection. Cyclin D1/CDK4 relocates to mitochondria at the same time as MnSOD enzymatic activation peaks without significant changes in total MnSOD protein level. The mitochondrial-localized CDK4 directly phosphorylates MnSOD at serine-106 (S106), causing enhanced MnSOD enzymatic activity and mitochondrial respiration. Expression of mitochondria-targeted dominant negative CDK4 or the MnSOD-S106 mutant reverses LDIR-induced mitochondrial enhancement and adaptive protection. The CDK4-mediated MnSOD activation and mitochondrial metabolism boost are also detected in skin tissues of mice receiving in vivo whole-body LDIR. These results demonstrate a unique CDK4-mediated mitochondrial communication that allows cells to sense environmental genotoxic stress and boost mitochondrial homeostasis by enhancing phosphorylation and activation of MnSOD.