A feature of urothelium is its capacity to self-repair. The Jack Birch Unit has shown that autocrine signalling mediated through the epidermal growth factor receptor is one of the main drivers of proliferation during regenerative repair.
One of the approaches used to study urothelial tissue regeneration is by introducing scratch wounds into confluent cultures and monitoring time to repair using time-lapse microscopy.
There is evidence that the immediate cellular response involves calcium signalling, which is utilized in a wide range of cellular processes, including cell migration and proliferation. Confocal microscopy and calcium imaging shows that immediately after wounding, cells at the leading edge of the wound show an elevated rise in calcium which is sustained for over 30 minutes, whereas cells distal to the wound show a transient rise.
Currently we are examining how the different calcium signal is translated into a differential response and are using computational agent-based modelling to help interpret how individual cell response contributes to the overall dynamics of the population.
In vitro-propagated normal human urothelial (NHU) cells may be induced to differentiate by a switch to medium containing serum (patent WO2004/011630). This results in the formation of a stratified urothelium that exhibits many of the differentiated and functional features of intact human urothelium. Electrophysiological and other functional parameters are used to assess urothelial barrier properties and to relate these to the differentiation characteristics of the in vitro-generated urothelium at the molecular level. This is a useful model for physiological studies of human urothelium.
The pathways involved in regulating proliferation and differentiation of human urothelium have been explored. We have shown that autocrine activation of the epidermal growth factor receptor (EGFR) is important in driving proliferation. When EGFR is blocked, activation of the PPARγ nuclear receptor will trigger a programme of gene expression changes that results in late/terminal urothelial cytodifferentiation.
Upon ligand-activation, PPARγ heterodimerises with the retinoid X receptor α (RXRα) to form a transcription factor complex that binds directly to peroxisome proliferator response elements (PPRE) within the promoters of target genes. PPARγ is phosphorylated on Ser84 by ERK2 and other MAP kinases, resulting in inhibition of PPARγ transcriptional activity. In NHU cell cultures, inhibition of EGF receptor signalling is therefore required for PPARγ to exist in its dephosphorylated “active” state.
In EGFR-inhibited NHU cells, activation of PPARγ results in the expression of specific genes/proteins associated with late/terminal urothelial differentiation including UPK1a, UPK2, UPK3a, CK20 and claudin3. PPARγ does not induce these differentiation-associated proteins directly, but via transactivation of PPARγ-regulated transcription factors including FOXA1 and IRF-1.
autocrine signalling - a form of cell signaling in which a cell secretes a hormone or chemical messenger (called the autocrine agent) that binds to autocrine receptors on that same cell, leading to changes in the cell.
Confocal microscopy - a powerful instrument that creates sharp images of fixed or living cells and tissues and can greatly increase optical resolution and contrast over that of a conventional microscope
stratified urothelium - An epithelial tissue comprised of more than one layer of epithelial cells