Prostaglandin E2 facilitates neurite outgrowth in a motor neuron-like cell line, NSC-34

Abstract

Prostaglandin E2 (PGE2) exerts various biological effects by binding to E-prostanoid receptors (EP1-4). Although recent studies have shown that PGE2 induces cell differentiation in some neuronal cells such as mouse DRG neurons and sensory neuron-like ND7/23 cells, it is unclear whether PGE2 plays a role in differentiation of motor neurons. In the present study, we investigated the mechanism of PGE2-induced differentiation of motor neurons using NSC-34, a mouse motor neuron-like cell line. Exposure of undifferentiated NSC-34 cells to PGE2 and butaprost, an EP2-selective agonist, resulted in a reduction of MTT reduction activity without increase the number of propidium iodide-positive cells and in an increase in the number of neurite-bearing cells. Sulprostone, an EP1/3 agonist, also significantly lowered MTT reduction activity by 20%; however, no increase in the number of neurite-bearing cells was observed within the concentration range tested. PGE2-induced neurite outgrowth was attenuated significantly in the presence of PF-0441848, an EP2-selective antagonist. Treatment of these cells with dibutyryl-cAMP increased the number of neurite-bearing cells with no effect on cell proliferation. These results suggest that PGE2 promotes neurite outgrowth and suppresses cell proliferation by activating the EP2 subtype, and that the cAMP-signaling pathway is involved in PGE2-induced differentiation of NSC-34 cells.

Generation of Cellular Reactive Oxygen Species by Activation of the EP2 Receptor Contributes to Prostaglandin E2-Induced Cytotoxicity in Motor Neuron-Like NSC-34 Cells

“…The role of caspase-3 activity in promoting neuronal differentiation was demonstrated across a broad spectrum of cell lineages including olfactory sensory neurons, neural stem cell, and PC12 cells [33]. Unlike these cells, previous studies of our and other laboratories revealed that the generation of cleaved caspase-3 is observed in undifferentiated NSC-34 cells as well as in differentiated forms [22,34]. Therefore, it seems that activation of caspase-3 is not responsible for the neuronal differentiation in NSC-34 cells.…”

“…The extent of cytotoxicity was quantified by measurement of lactate dehydrogenase (LDH) released into the medium during exposure to drugs. After NSC-34 cells had been incubated with PGE2 for 48 h, the supernatants were collected in new plates and LDH release was determined using a LDH-Cytotoxic Test kit (Wako Pure Chemical Industries) as described previously [22]. The absorbance of samples was measured at 570 nm using a microplate reader SH-1000Lab (Corona Electric).…”

“…This neuroprotective effect of butaprost was also conferred by cAMP analogs and was blocked by PKA inhibitors, suggesting that the neuroprotection afforded by EP2 activation is mediated through cAMP-dependent PKA activity in these cells [37]. Recently, we demonstrated that PGE2 promotes the conversion of undifferentiated NSC-34 cells to motor neuronal cells by activating the EP2 subtype, and that an exogenously applied cAMP analog, dbcAMP, facilitates neurite outgrowth with no effect on cell proliferation in undifferentiated NSC-34 cells [22]. Unlike the situation in undifferentiated cells, the results of the present study showed that dbcAMP partially mimicked PGE2-and butaprost-induced intracellular ROS generation and cell death in differentiated NSC-34 cells.…”

The Molecular Mechanisms Underlying Prostaglandin D2-Induced Neuritogenesis in Motor Neuron-Like NSC-34 Cells

“…PGE 2 promotes neuritogenesis in dorsal root ganglion neurons via EP2 (coupled to Gs protein) [14] and in sensory neuron-like ND7/23 cells via EP4 (coupled to Gs protein) [15]. Recently, our research has demonstrated that PGE 2 induces neurite outgrowth by activating EP2 in NSC-34 cells [16]. This suggests that PGE 2 is involved in neuritogenesis and the differentiation of various neurons including motor neurons.…”

“…Cultures were incubated at 37 • C and 5% CO 2 in a humidified atmosphere. For experimentation, NSC-34 cells were seeded onto Corning BioCoat™ Poly-L-Lysine TC-Treated Culture Dishes (Corning Inc., Corning, NY, USA) at a density of 12,500 cells/cm 2 and incubated for 24 h according to the method described previously [16].…”