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This Article Full Text Full Text (PDF) All Versions of this Article: JOE-07-0570v1 198/1/193    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Abidi, P. Articles by Azhar, S. PubMed PubMed Citation Articles by Abidi, P. Articles by Azhar, S.

¹ Department of Veterans Affairs Palo Alto Health Care System, Geriatric Research, Education and Clinical Center (GRECC), 3801 Miranda Avenue, Palo Alto, California 94304, USA² Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University, Stanford, California 94305, USA³ Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA

(Correspondence should be addressed to S Azhar at VA Palo Alto Health Care System; Email: salman.azhar{at}va.gov)

(S Leers-Sucheta is now at Pfizer Animal Health, Veterinary Medicine R & D, Pfizer Inc., 7000 Portage Road, Kalamazoo, Michigan 49001, USA)

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 superoxide-dismutase/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 Bt₂cAMP and Bt₂cAMP+hHDL₃-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 isoform, along with relatively low-level expression of p38 MAPK. By contrast, little or no expression was detected for p38 MAPKβ and p38 MAPK isoforms 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 or dnp38 MAPKβ construct also increased steroid hormone production; however, the effect was less pronounced after expression of either dnp38 MAPK or dnp38 MAPK construct. These results indicate that activated p38 MAPK mediates oxidant (excessive oxidative stress)-induced inhibition of adrenal steroidogenesis.