Oxidative stress-induced inhibition of adrenal steroidogenesis requires participation of p38 mitogen-activated protein kinase signaling pathway

Previous studies from this laboratory identified excessive oxidative stress as an important mediator of age-related decline in steroid hormone production. Here, we investigated whether oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. To accomplish these studies, we employed a highly responsive mouse adrenocortical cell line, Y1-BS1 cells that secrete large quantities of steroids when stimulated with lipoprotein plus hormone. Treatment of these cells with superoxide, H2O2 or 4-hydroxy-2-nonenal (HNE) significantly inhibited steroid production and increased phosphorylation and activation of p38 MAPK. None of the treatments altered the phosphorylation of either extracellular signal-regulated kinases or c-Jun N-terminal kinases (JNKs). Pretreatment of Y1-BS1 cells with MnTMPyP, a cell-permeable sup eroxide-disinutase/catalase mimetic reactive oxygen species (ROS scavenger), completely prevented the superoxide- and H2O2-mediated inhibition of steroid production. Likewise, antioxidant N-acetylcysteine completely blocked the HNE-induced loss of steroidogenic response. Incubation of Y1-BS1 cells with either MnTMPyP or NAC also upregulated Bt(2)cAMP and Bt(2)cAMP+hHDL(3)-stimulated steroid synthesis, indicating that endogenously produced ROS can inhibit steroidogenesis. Inhibition of p38 MAPK with SB203580 or SB202190 upregulated the basal steroid production and also prevented the oxidant-mediated inhibition of steroid production. mRNA measurements by qPCR indicated that Y1-BS1 adrenal cells predominantly express p38 MAPK alpha isoform, along with relatively low-level expression of p38 MAPK gamma. By contrast, little or no expression was detected for p38 MAPK beta and p38 MAPK delta isofoms in these cells. Transfection of Y1-BS1 cells with either caMKK3 or caMMK6 construct, the upstream p38 MAPK activators, decreased steroidogenesis, whereas transfection with dnMKK3 or dnMKK6 plasmid DNA increased steroidogenesis. Similarly, transfection of cells with a dnp38 MAPK alpha or dnP38 MAPK beta construct also increased steroid hormone production; however, the effect was less pronounced after expression of either dnp38 MAPK gamma or dnp38 MAPK delta construct. These results indicate that activated p38 NIAPK mediates oxidant (excessive oxidative stress)-induced inhibition of adrenal steroidogenesis.