Since its discovery in 2000, an increasing body of evidence has demonstrated the importance of the transient receptor potential vanilloid 4 (TRPV4) ion channel in regulating two critical components of endothelial function, namely the control of vascular tone and permeability. However, our understanding of both mechanisms is incomplete. TRPV 4 modulates vascular tone by activating endothelial nitric oxide and endothelial-derived hyperpolarization factor pathways. In this thesis, evidence is provided linking TRPV4 activation to the release of vasoactive prostaglandins. Pharmacological TRPV 4 activation elicited a striking release of prostaglandins both in vivo, in rats and in vitro from human umbilical vein endothelial cells (HUVECs). Prostaglandin release in vivo in rats contributed to the noted blood pressure reducing effect associated with the TRPV4 activator GSKIQ.16790A. Moreover, TRPV4-mediated prostanoid release in rats also appeared to counteract the lung permeability increase, assessed by lung wet weight, also associated with this agent. In subsequent experiments, the mechanism by which TRPV 4 mediates increases in vascular permeability were explored in vitro, using a HUVEC detachment assay. The results obtained demonstrate that two selective, structurally distinct TRPV4 activators, GSKI016790A and GSK634775, elicit divergent endothelial detachment responses in vitro, whereby both increase intracellular calcium levels but only GSKI016790A induced detachment. Surprisingly, the mechanism of GSKI016790A-mediated detachment was found to be independent of intracellular calcium or cytoskeletal modulation, but was mediated in pmt by activation of PKC and disruption of the plasma membrane. These findings demonstrate that TRPV 4 couples to prostaglandin-generating pathways in endothelial cells. The release of these prostanoids may play a role in modulating blood pressure and endothelial permeability, which may be of relevance in hypertensive states. In addition, these studies also highlight an intriguing differential coupling of TRPV 4 channels in isolated endothelial cells in response to structurally distinct agonists.

The epidermis is maintained through self-renewal of stem cells and differentiation of their progeny into interfollicular epidermis, hair follicles and sebaceous glands. In order to investigate the role of P-catenin in cell fate selection in adult epidermis I used a drug- regulated system to activate P-catenin signalling at specific stages of the hair cycle and for different lengths of time in the epidermis of transgenic mice. I found that following P-catenin activation resting hair follicles are recruited into the growth phase of the hair cycle, and new ectopic hair follicles form from existing hair follicles, interfollicular epidermis and sebaceous glands. The ectopic follicles grow in number and size to form trichofolliculomas. While a transient activation of p-catenin signalling is sufficient to trigger hair follicle morphogenesis, continuous activation is required to maintain hair follicle tumours, and titration of P-catenin signalling influences the quantity and timing of ectopic hair follicle formation. The new hair follicles form without major perturbation of the existing stem cell compartment, contain dermal papillae, undergo cycles of growth and regression, and express markers of the epidermal stem cell niche. Microarray analysis of the P-catenin induced genes suggested a role for the Hedgehog (Hh) and Notch pathways and vitamin D receptor downstream of P-catenin. I was able to investigate also the relationship between p > catenin and c-Myc in epidermal cell lineage choice. Finally, I developed an in vitro model for the analysis of lineage selection at the single cell level. I demonstrate the advantage of temporal, spatial and dosage control of p-catenin signalling to analyse the effects of its activation in adult epidermis and to investigate epidermal stem cell self-renewal and differentiation, an approach that could be applied more broadly to look at the interaction between multiple pathways in adult skin homeostasis.

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