Studies on cyclic AMP phosphodiesterases

[摘要] The second messenger cyclic AMP plays a pivotal role in the control of cell function through the transduction of extracellular signals into intracellular activation. However, this cyclic nucleotide negatively modulates the synthesis and release of inflammatory mediators and the proliferation of lymphocytes. The sole mechanism for degradation of cAMP is achieved through the action of cyclic AMP phosphodiesterses (PDEs). This activity is provided by a multigene family which produces a variety of enzymes. The PDE4 isoforms appear to be the predominant cAMP-metabolising enzymes in essentially all pro-inflammatory and immune cells implicated in the pathogenesis of asthma, arthritis and atopic dermatitis. With respect to the development of novel drugs to treat such diseases, an understanding of the regulation and the function of PDE4 enzymes has been identified as crucial. In this thesis, three different, but related, studies were carried out. The first study was an investigation of the expression and the regulation of cAMP phosphodiesterases in human Jurkat T lymphocytes. In the second study, the properties of the proteins encoded by five different mRNA transcripts from the human PDE4D gene were analysed after transient expression in COS7 cells. These included the PDE4D1 and PDE4D2 forms which I had shown to become induced in Jurkat T-cells with chronically elevated cAMP levels. Finally, the role of the N- and C-terminal regions of the inactive human PDE4A splice variant, 2EL originally cloned from a Jurkat T-cell library, was investigated. The Jurkat T-cell PDE activity was determined using selective PDE activators and inhibitors. Although a small amount of PDEl, PDE2 and PDE7 enzymes were present, PDE3 and PDE4 enzymes were shown to provide the major Jurkat T-cell PDE activity. Chronic elevation of inti'acellular cAMP levels achieved either by direct stimulation of adenylate cyclase or via stimulation of Gs or using a cell-permanent cAMP analogue increased PDE3 and PDE4 activities but did not affect PDE1, PDE2 and PDE7 activities. Despite the fact that the major PDE activity in quiescent Jurkat T-cells was contributed by PDE3 enzymes, PDE4 activity appeared to play a key role in the control of intracellular cAMP levels. Analyses done using RT-PCR and immunoblotting demonstrated that forskolin-elevated intracellular cAMP levels induced the 'short' form of PDE4D isoforms, PDE4D1 and PDE4D2, but did not induce any other PDE4 isoforms. However, chronic elevation of cAMP levels also led to the down-regulation of the novel 118 kDa PDE4A species found in resting Jurkat T-cells. The two human 'short' forms (PDE4D1 and PDE4D2) and the three 'long' forms (PDE4D3, PDE4D4 and PDE4D5) of human PDE4D isoforms were transiently expressed in COS7 cells. The 'short' PDE4D forms exhibited similar properties and were exclusively expressed in cytosol. In contrast, the 'long' forms showed different enzyme kinetics and were found in both particulate and cytosol fractions. It was suggested that the unique N-terminal regions of the PDE4D3, PDE4D4 and PDE4D5 proteins, which are derived from alternatively spliced regions of their mRNAs, are important in determining their enzyme activity, inhibitor sensitivity and subcellular localisation. The inactive human PDE4A species 2EL was cloned from resting Jurkat T-cells. However, it was not known that the inactivity is due to the premature truncation of either N- or C-terminal putative catalytic domain. This was investigated using chimeras and truncated PDE forms. It was shown that the C-terminal premature truncation terminates the 2EL's catalytic activity.

[发布机构] University:University of Glasgow

[关键词] Molecular biology