Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521725
Title: Serine phosphorylation of p47 PHOX in the regulation of NADPH oxidase activation in endothelial cells
Author: Teng, Lei
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2010
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
Endothelial cells express constitutively a NADPH oxidase 2 (Nox2), which by generating reactive oxygen species (ROS) plays an important role in the regulation of endothelial. function. The Nox2 has at least 4 regulatory subunits and p47phox is one of the major regulatory subunits of this enzyme. The p47pho )( has multiple serines at its C-terminal end, and the serine phosphorylation is essential for Nox2 activation. TNFa is an inflammatory cytokine with divergent biological functions in different cell types. A previous study has shown that TNFa is a stimulator of Nox2 activation in endothelial cells. However, it still remains to be elucidated whether p47phox serine phosphorylation is involved in TNFainduced endothelial ROS production. Therefore, the aim of this Ph.D project is to investigate the role of p47phox serine phosphorylation in TNFa-induced NADPH oxidase activation and endothelial ROS production. Ten serines ofp47phox (S303, S304, S315, S320, S328, S345, S348, S359, S370 and S379) were replaced by alanines in p47phox cDNA plasmid (pcDNA3.1 vector), using in vitro sitedirected mutagenesis. Mutated p47phox constructs were confirmed by molecular sequencing. A mouse lymphoid endothelial cell line (SVEC4-1 0) was used for gene transfection and the ROS production by SVEC4-10 cells was examined by NADPH-dependent lucigeninchemiluminescence. Under the basal condition (without TNFa stimulation), SVEC4-10 cells transfected with p47phox mutants showed no significant changes in ROS production. After acute TNFa (lOOU/ml, 30 minutes) stimulation, cells transfected with p47phox S303/304A, S315A, S328A, S345A, S370A or S379A plasmids showed a significant reduction in ROS production as compared to control cells transfected with vector only, whereas p47phox S320A, S348A and S359A mutations had no significant effect on TNFainduced ROS production. TNFa-induced MAPK phosphorylation was also examined in these cells transfected with p47phOX mutants. Comparing to cells transfected with pcDNA3.l vector, cells transfected with p47PhOX S328A, S345A, S370A or S379A plasmids reduced the levels of TNFainduced ERK1I2 phosphorylation, whereas cells transfected with p47phox S320A, S348A or S359A plasmids showed no significant changes in TNFa-induced ERK1I2 activation. Interestingly, p47phox S303/304A mutation reduced NADPH-dependent ROS production but had no significant effect on acute TNFa-induced ERK1I2, p38 MAPK and JNK activation. This was further verified using primary endothelial cells of mouse and human origin (mouse coronary microvascular endothelial cells and human lung endothelial cells), COS7phox cells and coronary microvascular endothelial cells isolated from p47Phox knockout mice. In conclusion, p47phox phosphorylation at multiple serines, including S303/304, S315, S328, S345, S370 and S379, is essential in TNFa-induced NADPH oxidase activation. Among these serines, serines 303 and 304 playa key role in acute TNFa.-induced ROS production, but are not involved in TNFa.-induced MAPK activation in cultured endothelial cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.521725  DOI: Not available
Share: