Phosphodiesterase 4 isoforms in macrophage development and function

Shepherd, Malcolm Cameron (2002) Phosphodiesterase 4 isoforms in macrophage development and function. PhD thesis, University of Glasgow.

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Phosphodiesterase 4 (PDE4) is a family of around 20 cyclic AMP (cAMP) hydrolysing enzymes. Expression of each isoform is regulated in a tissue and developmental specific manner. Such regulation suggests as yet undefined functions for each isoform. Understanding these functions will highlight likely therapeutic targets. I have employed various methods to identify possible functional roles for various PDE4 isoforms. PDE4B4 is a novel enzyme cloned from a rat brain cDNA library. By characterising the biochemical properties of this isoform in comparison to known, previously characterised members of the PDE4B family, I highlighted similarities and differences within this subfamily. Recombinant PDE4B4 was characterised in a COS-1 cell, temporary expression system. I demonstrated that PDE4B4 has a molecular weight lying between PDE4B1 and PDE4B2, is largely cytosolic, has a relatively low Km cAMP and is highly sensitive to inhibition by rolipram. As it has a UCR1 region it conforms to long form structure and is activated by PKA. It has an extreme N-terminal region homologous to PDE4D3 and behaves in a similar way in response to PKA phosphorylation. Development of macrophages from monocytes involves differential expression of various biochemical mediators. I developed a cell line model using the U937 pro-monocytic cell line and compared it against ex-vivo monocytes cultured in plastic. Using this model I identifies developmental changes in PDE4 isoform expression. PDE4A activity increased dramatically which was due in part to novel expression of PDE4A10. PDE4D isoform expression was entirely lost with no immunologically detectable enzyme present in macrophage like cells. PDE4B2 expression more than doubled in the mature cell. Loss of PDE4D and gain of PDE4B2 represents a shift from long to short form PDE4 dominance. I demonstrated a resultant switch in PDE4 response to extracellular signal related kinase (ERK) activation. Thus in monocytic cells EGF resulted in a decrease in total PDE4 activity, while in macrophage like cells PDE4 activity increased. To demonstrate a role for PDE4 in regulating macrophage function I stimulated the RAW 264.7-macrophage cell line with LPS in the presence of rolipram. I demonstrated a PGE2 dependent increase in iNOS expression and a PGE2 independent increase in COX2 expression. Such differential effects, suggests a compartmentalisation of rolipram's action. Inhibition of TNF? production was not dependent on PGE2 production. I then found that LPS activates PDE4 in an ERK dependent manner, but inhibits PDE3, highlighting further compartmentalisation of cAMP regulation. PDE4 activation was in part due to ERK dependent activation of the short form PDE4B2, downstream of LPS stimulation of RAW cells. Such activation may be associated with physical interaction between members of the ERK signalling cascade and PDE4B2. Next I demonstrated that crosstalk between ERK signalling and cAMP occurs in the opposite direction as rolipram leads to an early and elevated activation of ERK 1/2 in LPS stimulated RAW 264.7 macrophages. In an attempt to explain this effect I used rap 1 activation and PKA phosphorylation mutants, transfected into RAW cells to interfere with normal inflammatory signalling. No significant changes were observed in these studies. Finally I attempted to develop HIV-tat, PDE4 N-terminal, fusion proteins to inhibit individual PDE4 isoforms. I used primers encoding the HIV-tat peptide and PDE4 sequence to produce a cDNA fusion. This was cloned into a GST-expression vector and transformed into E-coli. Recombinant protein was expressed and purified using sepharose beads. Various problems were encountered in the course of this project, including the development of appropriate cloning primers and the production of proteins in inclusion bodies. The strategies employed to resolve these difficulties are discussed. Two further experiments are discussed. Firstly rolipram was found not to affect tritiated thymidine incorporation into proliferating HEK cells. This work argues against a role for PDE4 in regulating cell cycle in these cells. I also attempted to characterise PDE4 activity from the induced sputum of normal subjects. While PDE4 activity was found to survive the isolation process the intra-subject variability meant that useful interpretation of fluctuations based on therapeutic intervention would be impossible. In conclusion I have characterised a new member of the PDE4B family. It shares many characteristics with other members of the sub-family and long form PDE4 enzymes in general. I have shown upregulation of PDE4A10 and PDE4B2 in the maturation of macrophages and the loss of long form PDE4D3 and PDE4D5. This was found to have biochemical significance. PDE4B2 was shown to be important in the regulation of LPS activation of RAW cells and ERK/PDE4 crosstalk was found to occur in both directions. Rolipram was demonstrated to influence the behaviour of stimulated macrophages in a compartmentalised fashion, while LPS was found to activate PDE4 and inhibit PDE3. Finally I was unsuccessful in the development of a novel strategy for inhibiting individual PDE4 isoforms, by developing an HIV-tat fusion protein.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Housley, Prof. Miles and Thomson, Prof. Neil
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-71266
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 02 Jun 2021 13:36

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