title: Feedback inhibition of CREB signaling by p38 MAPK contributes to the negative regulation of steroidogenesis.
abstract: BACKGROUND
  Steroidogenesis is a complex, multi-steps biological process in which, cholesterol precursor is converted to steroids in a tissue specific and tropic hormone dependent manner. Given that steroidogenesis is achieved by coordinated functioning of multiple tissue specific enzymes, many steroids intermediates/metabolites are generated during this process. Both the steroid products as well as major lipoprotein cholesterol donor, high-density lipoprotein 3 (hHDL3) have the potential to negatively regulate steroidogenesis via increased oxidative stress/reactive oxygen species (ROS) generation.
  
  
  METHODS
  In the current study, we examined the effects of treatment of a mouse model of steroidogenesis, Y1-BS1 adrenocortical tumor cells with pregnenolone, 22(R)-Hydroxycholesterol [22(R)-diol] or hHDL3on ROS production, phosphorylation status of p38 MAPK and cAMP response element-binding protein (CREB), CREB transcriptional activity and mRNA expression of StAR, CPY11A1/P450scc and antioxidant enzymes, superoxide dismutases [Cu,ZnSOD (SOD1), MnSOD (SOD2)], catalase (CAT) and glutathione peroxidase 1 (GPX1). We also detected the steroid product in p38 MAPK inhibitor treated Y1 cells by HPLC-MS / MS.
  
  
  RESULTS
  Treatment of Y1 cells with H2O2greatly enhanced the phosphorylation of both p38 MAPK and CREB protein. Likewise, treatment of cells with pregnenolone, 22(R) diol or hHDL3increased ROS production measured with the oxidation-sensitive fluorescent probe 2',7'-Dichlorofluorescin diacetate (DCFH-DA). Under identical experimental conditions, treatment of cells with these agents also increased the phosphorylation of p38 MAPK and CREB. This increased CREB phosphorylation however, was associated with its decreased transcriptional activity. The stimulatory effects of pregnenolone, 22(R)-diol and hHDL3on CREB phosphorylation was abolished by a specific p38 MAPK inhibitor, SB203580. Pregnenolone, and 22(R) diol but not hHDL3upregulated the mRNA expression of SOD1, SOD2 and GPX1, while down-regulated the mRNA levels of StAR and CYP11A1. The p38 inhibitor SB203580 could increase the steroid production in HDL3, 22(R)-diol or pregnenolone treated cells.
  
  
  CONCLUSIONS
  Our data demonstrate induction of a ROS/p38 MAPK -mediated feedback inhibitory pathway by oxy-cholesterol and steroid intermediates and products attenuates steroidogenesis via inhibition of CREB transcriptional activity.
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 1 WenYuan Road, Nanjing, 210023, China.
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Li
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 1 WenYuan Road, Nanjing, 210023, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 1 WenYuan Road, Nanjing, 210023, China.
authors.forename: Zhuang
authors.initials: Z
authors.lastname: Ma
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 1 WenYuan Road, Nanjing, 210023, China.
authors.forename: Meina
authors.initials: M
authors.lastname: Wang
authors.affiliation: Geriatric Research, Education and Clinical Center, VA Palo Alto Health Care System, Palo Alto, CA, 94304, USA.|Stanford University School of Medicine, Palo Alto, CA, 94304, USA.
authors.forename: Wen-Jun
authors.initials: WJ
authors.lastname: Shen
authors.affiliation: Geriatric Research, Education and Clinical Center, VA Palo Alto Health Care System, Palo Alto, CA, 94304, USA.|Stanford University School of Medicine, Palo Alto, CA, 94304, USA.
authors.forename: Salman
authors.initials: S
authors.lastname: Azhar
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 1 WenYuan Road, Nanjing, 210023, China.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 1 WenYuan Road, Nanjing, 210023, China. huzg_2000@126.com.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Hu
mesh_terms: D000306:Adrenal Cortex Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000818:Animals; D015153:Blotting, Western; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D025461:Feedback, Physiological / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006888:Hydroxycholesterols / Q000494:pharmacology; D051379:Mice; D016877:Oxidants / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011284:Pregnenolone / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013256:Steroids / Q000096:biosynthesis*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D000094765:Glutathione Peroxidase GPX1; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D006888:Hydroxycholesterols; D016877:Oxidants; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; C003585:22-hydroxycholesterol; D011284:Pregnenolone; D006861:Hydrogen Peroxide; D002374:Catalase; D005979:Glutathione Peroxidase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D013482:Superoxide Dismutase; D048051:p38 Mitogen-Activated Protein Kinases; D000094765:Glutathione Peroxidase GPX1; C000722068:Gpx1 protein, mouse
keywords: CREB; Feedback regulation; Steroidogenesis; Steroids intermediates; p38 MAPK