UP REGULATION OF CYLOOXYGENASE-2 AND INDUCIBLE NITRIC OXIDE SYNTHASE IN MACROPHAGES DURING SELENIUM DEFICIENCY: ROLE OF NUCLEAR FACTOR-KAPPA B

Open Access
Author:
Zamamiri-Davis, Faith Alexandria
Graduate Program:
Pathobiology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
March 18, 2002
Committee Members:
  • Lorraine Sordillo Gandy, Committee Member
  • Benjamin Franklin Pugh, Committee Member
  • Madhu Reddy, Committee Chair
  • Andrew Thomas Henderson, Committee Member
Keywords:
  • inflammation
  • free radicals
  • transcription factor
  • macrophage
  • oxidative stress
  • selenium
Abstract:
While all cells produce reactive oxygen and nitrogen species (ROS/RNS) such as superoxide anion ( ), hydrogen peroxide (H2O2) nitric oxide (NO) and peroxynitrite (NOO-) during normal respiration, activated macrophages produce elevated levels of these species as components of the immune respiratory burst. In addition, ROS/RNS are important members of cellular signaling pathways, particularly those leading to the inducible activation of nuclear factor-kappa B (NF-êB). NF-êB is a redox sensitive transcription factor, which is associated with the expression of various immediate-early genes including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The products of these enzymes, prostaglandins and NO, together with the acute phase cytokines regulate the initiation and duration of the inflammatory response. Thus, the excess production of ROS/RNS by macrophages may contribute significantly to the pathophysiology of inflammatory disorders if it is not carefully controlled by cellular antioxidant defense systems. To further explore the role of selenium (Se) status in macrophage immune function, the RAW 264.7 macrophage cell line was cultured in media deficient of or supplemented with sodium selenite. The activity of Se –dependent glutathione peroxidase (Se-GPx) was monitored to confirm deficiency. At least an 8-fold difference in the enzyme activity was confirmed and maintained throughout several passages. Fluorescent labeling and flow cytometric analysis confirmed a higher degree of oxidative stress in the Se deficient group prior to any stimulus. Following stimulation with lipopolysaccharide, Se deficient macrophages were shown to express higher levels of the key inflammatory enzymes, COX-2 and iNOS as assessed by mRNA, protein and enzyme activity. To determine the role of NF-êB in the over expression of these enzymes, it was assessed at the level of nuclear translocation, binding and initiation of transcription. At three levels were NF-êB was up regulated in the Se-deficient macrophages as compared to the Se-supplemented cells, suggesting that it is involved in the up regulation of key enzymes associated with inflammatory response. To confirm NF-êB involvement, luciferase reporter constructs for COX-2 and iNOS were generated and assessed via transient transfection in both Se-deficient and Se-supplemented group. Deletion or truncation of NF-êB binding sites from the promoters resulted in significant decreases in luciferase activity. Furthermore, upstream mediators, MEKK1, NIK, IKKb and IêBa were detected at higher levels in the Se-deficient macrophages. A greater degree of serine phosphorylation of MEKK1 and also serine and tyrosine phosphorylation of IêBa was observed in deficient conditions, as well. When the Se deficient macrophages were repleted with Se, restoration of Se-GPx activity was observed, along with decreased levels of MEKK-1, p-IêBa and COX-2, comparable to that of the Se-supplemented cultures. In conclusion, these results suggest a role for Se in the regulation of NF-kB and subsequent expression of COX-2 and iNOS in a macrophage cell line. While the exact target or mechanism of Se regulation has not been fully identified in this model, the data suggest that it may be at the level of kinases upstream of NF-êB, notably MEKK1. These findings support the data of other studies in this field and highlight the importance of Se status in cellular redox signaling.