cyclic-gmp and genipin

Genipin, a herbal iridoid, is known to have both neuroprotective and neuritogenic activity in neuronal cell lines. As it is structurally similar to tetrahydrobiopterin, its activity is believed to be nitric oxide (NO)-dependent. We previously proposed a novel neuroprotective activity of a genipin derivative, (1R)-isoPropyloxygenipin (IPRG001), whereby it reduces oxidative stress in RGC-5, a neuronal precursor cell line of retinal origin through protein S-nitrosylation. In the present study, we investigated another neuritogenic property of IPRG001 in RGC-5 cells and retinal explant culture where in we focused on the NO-cGMP-dependent and protein S-nitrosylation pathways. IPRG001 stimulated neurite outgrowth in RGC-5 cells and retinal explant culture through NO-dependent signaling, but not NO-dependent cGMP signaling. Neurite outgrowth with IPRG001 requires retinoic acid receptor β (RARβ) expression, which is suppressed by an RAR blocking agent and siRNA inhibition. Thereby, we hypothesized that RARβ expression is mediated by protein S-nitrosylation. S-nitrosylation of histone deacetylase 2 is a key mechanism in chromatin remodeling leading to transcriptional gene activation. We found a parallelism between S-nitrosylation of histone diacetylase 2 and the induction of RARβ expression with IPRG001 treatment. The both neuroprotective and neuritogenic activities of genipin could be a new target for the regeneration of retinal ganglion cells after glaucomatous conditions.

Topics: Analysis of Variance; Animals; Cell Line, Transformed; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Histone Deacetylase 2; Humans; Iridoid Glycosides; Iridoids; Male; Neurites; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Receptors, Retinoic Acid; Retina; Retinal Ganglion Cells; RNA, Small Interfering; Signal Transduction

We have reported previously that genipin, a natural iridoid compound, induces neuritogenesis through a nitric oxide (NO)-cyclic GMP (cGMP)-cGMP-dependent protein kinase (PKG) signaling pathway in PC12h cells and that neuronal NO synthase (nNOS) is one of the target molecules of genipin in vitro. Recently, it has been suggested that the neurotrophic effects of NO are due to its direct activation of receptor-tyrosine kinase, especially TrkA. In this study, we investigated whether mouse neuroblastoma Neuro2a cells, which express nNOS but not TrkA, respond to genipin with neurite outgrowth through the mechanism observed in PC12h cells, to assess the involvement of TrkA in the mechanism. Neuro2a cells expressed all three types of NO synthase (NOS), and nNOS was detectable as the main component in Western blot analysis. Genipin significantly induced neurite outgrowth and activation of NADPH-diaphorase, which were significantly blocked by a non-selective NOS inhibitor. Both a soluble guanylate cyclase inhibitor and a PKG inhibitor also inhibited the genipin-induced neuritogenesis. Genipin induced sustained phosphorylation of mitogen-activated protein kinase (MAPK). In fact, the genipin-induced neurite outgrowth was completely inhibited by a specific MAPK kinase inhibitor. Moreover, a NOS inhibitor abolished MAPK phosphorylation as well as neurite outgrowth in genipin-treated cells. These results suggest that genipin induces neurite outgrowth through an NO-cGMP-PKG signaling pathway followed by MAPK phosphorylation without TrkA activation in Neuro2a cells and that PKG downstream to NOSs, which may be mainly nNOS, is very important for the signaling molecule to induce neuritogenesis by genipin.

Topics: Animals; Cell Line, Tumor; Cholagogues and Choleretics; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Iridoid Glycosides; Iridoids; Mice; Mitogen-Activated Protein Kinases; NADPH Dehydrogenase; Neurites; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Phosphorylation; Signal Transduction

We have demonstrated previously that a natural iridoid compound, genipin, induced neuritogenesis through activation of nitric oxide synthase (NOS) and mitogen-activated protein kinase (MAPK) in PC12h cells. In this paper, we investigated whether cyclic GMP (cGMP) and cGMP-dependent protein kinase (PKG) are involved in the neuritogenesis as a result of NOS activation. Furthermore, we also investigated the relationship between cGMP and MAPK activation in the signaling pathway. The genipin-induced neuritogenesis accompanied by induction of neurofilament was significantly inhibited by 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) and KT5823, inhibitors of soluble guanylate cyclase and PKG, respectively. Genipin-induced MAPK phosphorylation was also abolished by ODQ. These inhibitory effects of ODQ were similar to those observed for nerve growth factor (NGF)-induced neurite outgrowth and MAPK phosphorylation. The membrane-permeable cGMP analog, 8-Bromo-cGMP, had prominent neuritogenic activity, which was completely inhibited by a MAPK kinase inhibitor, PD98059. These results suggest that the soluble guanylate cyclase-PKG signaling pathway is important for MAPK activation by genipin as well as NGF during neuritogenesis in PC12h cells.

Topics: Animals; Blotting, Western; Cell Differentiation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Guanylate Cyclase; Iridoid Glycosides; Iridoids; Mitogen-Activated Protein Kinases; Nerve Growth Factors; Neurites; Neurofilament Proteins; Nitric Oxide; PC12 Cells; Phosphorylation; Pyrans; Rats; Signal Transduction