Initiation and termination of cAMP signalling in PANC-1 cells: interplay between cAMP and Ca2+ signalling cascades.

Parker, Tony Initiation and termination of cAMP signalling in PANC-1 cells: interplay between cAMP and Ca2+ signalling cascades. PhD thesis, University of Liverpool.

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is noted for its resistance to therapy and poor prognosis. PDAC is characterised by extensive local invasion and metastases at distant organs. Having previously shown that the cAMP cascade had regulatory effects on cell migration/invasion in PDAC cells, which are vital processes in metastasis formation, we decided to characterise the cAMP signalling machinery in PANC-1 cells. In this study we adopted a live-cell imaging approach taking advantage of genetically engineered probes that use Fӧrster resonance energy transfer (FRET) to reveal cAMP concentration and PKA activity at a single cell level. Using the Epac-based cAMP sensor H134 we found that PDE3 and PDE4 are the principal cAMP destroyers, whereas β-adrenoceptors are the main physiological cAMP-cascade activators, in PANC-1 cells. Downstream, using Boyden chamber assays we found that PDE3 has the biggest role in cell migration under ‘basal’ conditions, whereas PDE4 has a bigger role in the presence of isoproterenol. However, isoproterenol on its own did not influence PANC-1 cell migration despite having the ability to increase cAMP concentration inside the cell. This part of the study puts forward PDE3 and PDE4 as potential targets for reducing migration/invasion of PDAC. In the second part of the study, we found that these cells have an efficient Ca2+ signalling system; in which ORAI1 is the main mediator of store-operated Ca2+ entry (SOCE) in PANC-1 cells. To explore the possibility of a Ca2+-cAMP crosstalk in PANC-1 cells, we used the AKAR4 probe to measure PKA activity during Ca2+ responses. The application of neurotensin (NT), a well-known IP3-producing agonist in this cell type, induced a Ca2+ response which was accompanied by an increase in PKA activity. SOCE is likely to play an important role in this process since the activation of SOCE by thapsigargin-mediated store depletion consistently and robustly increased PKA activity. To further investigate the downstream roles of PKA signalling in PANC-1 cells we utilised immunofluorescence to visualise the distribution of PKA activity. Using antibodies specific for phosphorylated PKA substrates, we found that phosphorylated PKA substrates are preferentially concentrated in the nucleus; and notably at the leading edges of PANC-1 cells displaying a migratory phenotype. At the leading edges phosphorylated PKA substrates colocalised with actin-rich ruffles. Interestingly, utilising the rapamycin-inducible heterodimerisation system to reveal endogenous ER-PM junctions in migrating PANC-1 cells, we found that ER-PM junctions are also situated in close proximity to the leading edge. These results suggest that SOCE activates PKA responses in the region strategically important for PDAC cell migration.

Item Type:	Thesis (PhD)
Additional Information:	Date: 2015-01 (completed)
Subjects:	?? QP ??
Depositing User:	Symplectic Admin
Date Deposited:	15 Sep 2015 09:30
Last Modified:	17 Dec 2022 01:33
DOI:	10.17638/02011139
URI:	https://livrepository.liverpool.ac.uk/id/eprint/2011139