Physiological roles of neuregulin-1/ErbB signaling in autonomic gangalia
Dept. of Medicine/박사
Neuregulin (NRG) is a growth factor which binds to the ErbB family of receptor tyrosine kinase and is expressed in the central and peripheral neurons to regulate various functions including development, cell survival, and gene expression. However, little is known about physiological significance of NRG in adult autonomic neurons. Among different products of the NRG genes, NRG1 is known to contain acetylcholine receptor inducing activity (so called ARIA) and thus regulate expression of nicotinic acetylcholine receptor (nAChR) at central synapse and neuromuscular junctions. The purpose of this study is to examine the expression, release, and function of NRG1 in autonomic major pelvic ganglia (MPG) containing sympathetic and parasympathetic neurons. First of all, quantitative RT-PCR revealed that MPG neurons endogenously express Type I and Type III NRG1, and ErbB2/ErbB3 with lack of ErbB4. In addition, the form is more dominant than the form for EGF-like domain in MPG neurons. To evoke release of the endogenous NRG1 from MPG, the preganglionic nerves were electrically stimulated using suction electrodes. Immediately after stimulation, MPG were isolated and transferred to culture media. Western blotting of the conditioned media revealed soluble NRGs (~48 kDa) which were cleaved from pro-NRG1. Consistent with this, high potassium (50 mM)-induced depolarization elicited release of NRG1 from MPG. Moreover neurotrophic factors including NGF, BDNF, and CNTF also stimulated release of NRG1 from MPG. Release of the soluble NRG1 was also proven by detecting the phosphorylation of the ErbB2 receptors in the electrically stimulated MPG neurons. Maximal phosphorylation of ErbB2 receptors was induced within 10 min in MPG neurons. Patch-clamp experiments showed that nicotinic acetylcholine receptor (nAChR) currents were significantly enhanced in both types of the MPG neurons
incubated for 6 hr in the conditioned media. Taken together, this study suggest that NRG1 can function as an endogenous regulator of nAChRs in autonomic MPG neuronsWhen MPG neurons were incubated with the recombinant NRG1 (5 nM) for 6 hr in cultured, nAChRs 3 and 4 subunits were 1.8-fold and 1.5-fold increased, respectively as revealed using real-time PCR and Western blotting analyses. Consistent with the molecular data, patch-clamp studies showed that NRG1 significantly enhances nAChR currents in both types of the MPG neurons. In contrast, NRG1 failed to alter voltage-gated T- and N-type calcium currents. Unlike NRG1, other trophic factors (NRG1, SMDF, NGF, BDNF, and CNTF) had no effects on nAChR currents. Cycloheximide (10 g/ml) negated the effects of NRG1 on the nAChR current. Furthermore, non-genomic effect of NRG1 on nAChR channels was not observerd. NRG1-induced increase in nAChR currents was blocked by AG825, an ErbB2 inhibitor and genistein, a non-specific tyrosine kinase inhibitor. LY294002 and wortmannin, selective PI3K inhibitors or U0126, a selective MEK inhibitor abolished completely the NRG1-induced increase of nAChR current. Consistent with these data, the PI3K inhibitors (LY294002 and wortmannin) and the MEK inhibitor (U0126) negated NRG1-induced phosphorylation of Akt and ErK1/2, respectively. Interestingly, the PI3K inhibtors prevented the increase in pErk1/2 levels in NRG1-treated MPG neurons similar to U0126. However, the MEK inhibitor did not affect NRG1-induced phosphorylation of Akt.In conclusion, NRG1 can be physiologically released by electrical activity and function as ARIA via the PI3K-MEK signalling cascade and thus may play a role in regulation of excitatory synaptic transmission in autonomic MPG.