Thesis

The regulation of nuclear factor kappa B (NFkB) activation mediated by proteinase-activated receptor-2 (PAR-2)

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Awarding institution
  • University of Strathclyde
Date of award
  • 2006
Thesis identifier
  • T11672
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Abstract
  • Proteinase-activated receptor-2 (PAR-2) is the second member of the new subfamily of G-protein coupled receptors with a novel mode of mechanism of activation. As with other family members, PAR-2 is activated through the proteolytic cleavage of the amino terminal of the receptor, thus un-masking a tethered ligand which then binds to the receptor causing intramolecular activation. Serine proteases such as trypsin and tryptase serve as the predominant endogenous activators for PAR-2, whilst synthetic activating peptides derived from the tethered ligand sequence are also able to stimulate receptor activation without proteolysis. The expression of PAR-2 has been detected in a variety of human tissues and receptor activation has been shown to mediate diverse biological functions including haemostasis and inflammation. However, the signalling mechanisms underlying PAR-2-induced cellular effects remain largely undefined. Thus this study sought to determine the signalling events following PAR-2 stimulation in NCTC-2544 skin cell line stably expressing PAR-2 and normal human epidermal keratinocytes, in particular the activation of NFкB. Initially PAR-2 was demonstrated to activate NFкB at the levels of IкBα loss, p65 NFкB phosphorylation, DNA binding and transcriptional activation. PAR-2 was also found to stimulate the three major MAP kinases, namely ERK, p38 MAPK and JNK. In addition, this study has included the work to examine the effects of putative PAR-2 inhibitors; K-14585 and ENMD-1068. K-14585 was a weak antagonist in some assay systems but not others, whilst ENMD-1068 was ineffective. The intermediates upstream in the NFYB pathway stimulated by PAR-2 were assessed. Phosphorylation of p65 NFкB was found to be dependent upon Gαq/₁₁, PKC isoforms, calcium mobilisation and IKKP. Interestingly, the inhibition of Gαq/₁₁, activity did not affect NFкB-DNA binding but partially suppressed NFкB-driven transcription. These findings point to the differential regulations of these intermediary components on different levels of NFкB activation mediated by PAR-2.
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DOI
EThOS ID
  • uk.bl.ethos.436099
Date Created
  • 2006
Former identifier
  • 739745

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