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Document Type:Latin Dissertation
Language of Document:English
Record Number:52910
Doc. No:TL22864
Call number:‭U229352‬
Main Entry:David A. Medina-Tato
Title & Author:Role of phosphoinositide 3-kinase in tnf[alpha] signalling in a549 lung alveolar cellsDavid A. Medina-Tato
College:University of Bath (United Kingdom)
Date:2007
Degree:Ph.D.
student score:2007
Abstract:Tumour necrosis factor (TNF) is a pleiotropic cytokine essential to the immune response within chronic inflammatory respiratory diseases. In lung parenchymal cells, TNF induces the upregulation of cytokines, chemokines, adhesion molecules and inflammatory mediators, which are all important molecules within the immune response of the lung. The role of individual phosphoinositide 3-kinase (PI3K) isoforms within these processes is unknown at present. After carrying out initial experiments to determine the effect of PI3K inhibitors on a primary murine lung preparation, A549 lung epithelial cells were chosen as an adequate model for subsequent experimentation. Small interfering (si) RNA technology was employed to explore the role of specific PI3K isoforms. After validation of this technique through mRNA and protein quantitation, the impact of siRNA-mediated PI3K silencing on basic cell functions was analysed by determining the ability of A549 cells to heal wounds and survive under basal conditions. During the course of these experiments, PI3K class 11- siRNA treated cells were unable to survive; while class 11- and p110-siRNA treated cells possessed a delayed wound healing response. Using PI3K isoform-specific inhibitors and siRNA on A549 cells, the role of PI3K isoforms in TNF-induced cell signalling and functions was then determined. Under TNF-stimulated conditions, p110-specific inhibition, by siRNA or by the p110-specific inhibitor IC87114, decreased the levels of cyclooxygenase (COX)-2 and its consequent prostaglandin (PG) E<sub>2</sub> upregulation. Studies into the phosphorylation levels of different transcription factors thought to be active during COX-2 production, led to the determination that p110 activity is also important in cJun activation, an important promoter of COX-2 gene expression. Furthermore, using a specific peptide inhibitor of cJun amino-terminal kinase, it was determined that cJun activity is important for TNF-induced COX-2 and PGE2 upregulation. Mitogen activated protein kinase signalling (extracellular signal-regulated kinase and cJun N-terminal kinase) was not affected by PI3K inhibition, while Class I p110-specific siRNA or a p110/p110 inhibitor TGX-121 donwregulated Akt (a downstream mediator of PI3K activity) phosphorylation induced by TNF. IC87114 or siRNA specific for p110 or p110, had no effect in Akt activation. With regards to other cell functions, ICAM-1 expression was not affected by PI3K inhibition, but proved to be dependent on mammalian target of rapamycin (mTOR) activity; while interleukin-8 upregulation was PI3K- and mTOR-independent. The findings herein described suggest that class IA p110 is the main isoform involved in TNF-induced Akt phosphorylation and that p110 is an important regulator of TNF-induced cJun activation and COX-2 upregulation.
Subject:Psychology--Abstracting, Bibliographies, Statistics; DXN113864; Pure sciences; Biochemistry; 0487:Biochemistry
Added Entry:University of Bath (United Kingdom)