3 research outputs found
Dissecting the roles of the Dectin-1R in the bladder innate defences
Ph. D. ThesisPrevious work using immortalised cells transfected with a NF-κB reporter and challenged with
zymosan, to mimic a fungal infection, suggested possible co-operation between Dectin-1 and
TLR5 receptors in urogenital tissues. These data were interesting as research describing
Dectin-1R functioning relates specifically to myeloid derived cells and TLR5, classically, is
known to function as a homodimer. The aim of the research reported in this thesis was
therefore to focus on the Dectin-1 and TLR5 receptors in the bladder urothelium, with the
objective of investigating Dectin-1 receptor functioning, Dectin1/TLR5 receptor co-operation
and cell signalling events following a zymosan (β-glucan) challenge.
Western analyses of cell lysates from a bladder biopsy and immortalised RT4 bladder cells
using a Dectin-1R antibody (R&D systems, AF1859) revealed the synthesis of proteins
representing full-length and truncated (stalk-less) Dectin-1 receptors. These data supported
the use of the immortalised RT4 cells in all subsequent analyses exploring Dectin-1R
functioning in the urothelial tissues. Following challenge of the cells with the yeast cell wall
extract zymosan (50ug/ml) increased synthesis of an array of host defence agents including
hBD2, IL-8 and LCN2 were observed (p<0.05). Furthermore IL-8 effector synthesis was
significantly reduced following CLEC7A (Dectin-1R) gene knockdown (p<0.05) and antibody
blocking of the Dectin-1R (p<0.01). These data supported synthesis of the Dectin-1R in
urothelial cells and a role in defending the bladder against a fungal challenge.
Knocking-down the TLR5 gene in RT4 cells (80% knockdown) and challenging the cells with
zymosan, resulted in a significant reduction in hBD2, IL8 and LCN2 synthesis (p<0.05). These
data suggested co-operation between the Dectin-1 and TLR5 receptors in bladder cells, which
was supported by immunostaining and a proximity ligation assay approach. In addition these
analyses supported clustering of the Dectin-1 and TLR5 receptors following activation.
Experiments to explore roles for the encoded Dectin-1 receptor isoforms, 1, 2 and 4, in the
TLR5 co-operation events involved engineering a suite of stable cell lines each over-expressing
a Dectin-1 receptor isoform and either a TLR5 full-length or TLR5 truncated receptor. This
approach exploited the pVitro2-neo-mcs vector, which is able to co-express two cDNA
sequences simultaneously. However, the resultant cell lines did not over-express the Dectin-1
and TLR5 receptors. It was hypothesised that the increased number of receptors synthesised
by the cells over-loaded the urothelial cell membranes causing gene silencing and/or cell death.
The signalling mechanisms activating the transcription of host effector genes following a
zymosan challenge of RT4 bladder cells were explored using western analyses and an antibody
to Syk-P (Cell Signalling, C87C1), SYK gene knockdown and piceatannol (100 ug/ml) a Syk
inhibitor followed by ELISA to measure LCN2 and IL-8 media concentrations. No
phosphorylation of Syk was observed and the knock-down/inhibition approaches had no
significant effects on the media concentrations of either IL-8 or LCN2. However, knockdown of
the RAF1 gene resulted in decreased secretion of the host defence molecules LCN2 and IL-8
(p<0.01). Additionally western blot analyses showed phosphorylation and degradation of the
NF-κB inhibitor IκBα. These data suggested that Dectin-1R activation in response to zymosan in
bladder cells involved Raf-1 signalling, which supported a non-canonical signalling pathway.
In summary, Dectin-1 receptors were shown to be synthesized in RT4 bladder epithelial cells
and were activated in response to zymosan (fungal) challenge resulting in the production of
host defence effector molecules. Data showed potential co-operation between Dectin-1 and
TLR5 receptors in mediating the bladder cell response. Dectin-1R signalling in urothelial cells
was also orchestrated via a non-canonical signalling pathway involving Raf-1. These data
support a novel host defence mechanism, involving Dectin-1 and TLR5 receptors functioning
co-operatively, to defend urothelial from fungal infections.Dr William Edmund Harker Foundatio
Reconstruction and signal propagation analysis of the Syk signaling network in breast cancer cells
International audienceThe ability to build in-depth cell signaling networks from vast experimental data is a key objective of computational biology. The spleen tyrosine kinase (Syk) protein, a well-characterized key player in immune cell signaling, was surprisingly first shown by our group to exhibit an onco-suppressive function in mammary epithelial cells and corroborated by many other studies, but the molecular mechanisms of this function remain largely unsolved. Based on existing proteomic data, we report here the generation of an interaction-based network of signaling pathways controlled by Syk in breast cancer cells. Pathway enrichment of the Syk targets previously identified by quantitative phospho-proteomics indicated that Syk is engaged in cell adhesion, motility, growth and death. Using the components and interactions of these pathways, we bootstrapped the reconstruction of a comprehensive network covering Syk signaling in breast cancer cells. To generate in silico hypotheses on Syk signaling propagation, we developed a method allowing to rank paths between Syk and its targets. We first annotated the network according to experimental datasets. We then combined shortest path computation with random walk processes to estimate the importance of individual interactions and selected biologically relevant pathways in the network. Molecular and cell biology experiments allowed to distinguish candidate mechanisms that underlie the impact of Syk on the regulation of cortactin and ezrin, both involved in actin-mediated cell adhesion and motility. The Syk network was further completed with the results of our biological validation experiments. The resulting Syk signaling sub-networks can be explored via an online visualization platform