Neurosteroidogenesis has not been well elucidated due to the very low level of steroidogenic proteins in the brain. Here we report the first demonstration of the neuronal localization of neurosteroidogenic systems as well as the regulation of neurosteroidogenic activity in the adult rat hippocampus. Significant localization of cytochrome P450scc was observed in pyramidal neurons and granule neurons by means of immunohistochemical staining of slices. We also observed the colocalization, in hippocampal neurons, of P450scc with redox partners, hydroxysteroid sulfotransferase and steroidogenic acute regulatory protein. The distributions of astroglial cells and oligodendroglial cells showed very different patterns from that of the P450scc-containing cells. The expression of P450scc, redox partners, the sulfotransferase, and steroidogenic acute regulatory protein was also confirmed by Western blot analysis. The process of active neurosteroidogenesis was stimulated by exposing neurons to N-methyl-D-aspartate. Upon stimulation with N-methyl-D-aspartate, Ca(2+) influx through the N-methyl-D-aspartate subtype of glutamate receptors occurred, and significant net production of pregnenolone and pregnenolone sulfate was observed in the hippocampus. This neurosteroid production was considerably suppressed by the addition of antagonists of N-methyl-D-aspartate receptors, by Ca(2+) depletion, or by the addition of an inhibitor of P450scc. Upon stimulation with N-methyl-D-aspartate, the processing of full-length steroidogenic acute regulatory protein (37-kDa) to the truncated 30-kDa steroidogenic acute regulatory protein was observed. Taken together, these observations imply that hippocampal neurons synthesize neurosteroids. This synthesis may be stimulated and regulated by glutamate-mediated synaptic communication.

No abstract

Extracellular superoxide dismutase (EC-SOD) is one of the main SOD isozymes and plays an important role in the prevention of cardiovascular diseases by accelerating the dismutation reaction of superoxide. Royal jelly includes 10-hydroxy-2-decenoic acid (10H2DA, 2), which regulates the expression of various types of genes in epigenetics through the effects of histone deacetylase (HDAC) antagonism. The expression of EC-SOD was previously reported to be regulated epigenetically through histone acetylation in THP-1 cells. Therefore, we herein evaluated the effects of the royal jelly constituents 10-hydroxydecanoic acid (10HDA, 1), sebacic acid (SA, 3), and 4-hydroperoxy-2-decenoic acid ethyl ester (4-HPO-DAEE, 4), which is a derivative of 2, on the expression of EC-SOD in THP-1 cells. The treatment with 1 mM 1, 2, or 3 or 100 μM 4 increased EC-SOD expression and histone H3 and H4 acetylation levels. Moreover, the enrichment of acetylated histone H4 was observed in the proximal promoter region of EC-SOD and was caused by the partial promotion of ERK phosphorylation (only 4) and inhibition of HDAC activities, but not by the expression of HDACs. Overall, 4 exerted stronger effects than 1, 2, or 3 and has potential as a candidate or lead compound against atherosclerosis.

Leukemic cell lines, such as U937, THP-1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro. It is well known that TPA enhances reactive oxygen species (ROS) generation through the activation of NADPH oxidase (NOX), and ROS act as mediators in TPA signaling. Extracellular-superoxide dismutase (EC-SOD) is a major anti-oxidative enzyme that protects the cells from damaging effects of superoxide. Recently, the reduction of Cu/Zn-SOD and the induction of Mn-SOD by TPA in leukemic cells have been reported; however, the regulation of EC-SOD by TPA remains poorly understood. Here, we explored the regulation of EC-SOD during the monocytic differentiation of U937 cells by TPA. We observed the reduction of EC-SOD and Cu/Zn-SOD, whereas the induction of Mn-SOD during the differentiation of U937 cells. The reduction of EC-SOD and Cu/Zn-SOD was attenuated by pretreatments with GF109203X (an inhibitor of protein kinase C, PKC), diphenyleneiodonium (an inhibitor of NOX), and U0126 (an inhibitor of mitogen-activated protein kinase kinase, MEK/extracellular-signal regulated kinase, ERK). Interestingly, pretreatment with BAY11-7082 (an inhibitor of nuclear factor-κB, NF-κB) suppressed the reduction of Cu/Zn-SOD, but not of EC-SOD. Furthermore, we also determined the involvement of newly synthesized protein and the instability of mRNA in the reduction of EC-SOD. Overall, our results suggest that the expression of EC-SOD is decreased by TPA through intracellular signaling consisting of PKC, NOX-derived ROS and MEK/ERK, but not of NF-κB signaling.

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall. EC-SOD is also observed in monocytes/macrophages, and its high expression contributes to the suppression of atherosclerosis by scavenging superoxide. The molecular mechanisms governing cell-specific expression of EC-SOD are mostly unknown, while the anti-oxidative effect of EC-SOD is well recognized. In this study, we investigated the expression of EC-SOD during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of THP-1 cells, which is not expressing its gene in the basal phase. We confirmed the significant induction of EC-SOD in a TPA time-dependent manner, and that induction was completely blocked by pre-treatment with GF109203X, an inhibitor of protein kinase C, U0126 and PD98059, inhibitors of mitogen-activated protein kinase kinase/extracellular-signal regulated kinase. Moreover, we determined the involvement of NADPH oxidase-derived reactive oxygen species in that induction. Overall, we considered that these results may contribute to clarify the cell-specific expression of EC-SOD.