Ultraviolet radiation (UVR) evokes a plethora of effects in skin ranging from erythema and epidermal apoptosis, to photoageing and ultimately photocarcinogenesis. UVR is also used successfully as a therapy for atopic eczema and psoriasis and both the beneficial and detrimental effects of UVR depend partly on wavelength of radiation. Understanding the signaling mechanisms activated in human skin and the wavelength specificity, may provide insight into the molecular mechanisms regulating the pathological and therapeutic actions of UVR. Nuclear factor of activated T-cells (NFAT) is a calcium/calcineurin-regulated transcription factor that resides in an inactive, phosphorylated state in the cytoplasm. Dephosphorylation of NFAT by calcineurin evokes nuclear translocation and transcriptional activation of NFAT. It is shown in this thesis that UV-induced NFAT nuclear translocation and transcriptional activation is wavelength dependent. Five different UVR sources, covering the UV spectrum, each evoked a dose-dependent increase in NFAT transcriptional activity. The action spectrum for NFAT activation was derived by mathematical induction which highlights an inverse association between wavelength and NFAT transcriptional activity. UV-induced translocation of endogenous NFAT2 and GFP-NFAT2 is also wavelength-dependent and it appears that UVR specifically activates NFAT2,in HaCaT keratinocytes. For the first time, it is also demonstrated that UVR stimulates nuclear translocation of NFAT2 in skin equivalent models and in human skin in vivo. Cyclooxygenase-2 (COX-2) is a proinflammatory, procarcinogenic enzyme that has been implicated in transmitting the procarcinogenic effects of UVR. COX-2 luciferase reporter vec~ors which lacked functional NFAT binding sites, indicate that NFAT is required for maximal induction of the COX-2 promoter by UVR. Induction of COX-2 protein in HaCaTs by UVR is reduced by inhibiting UV-induced NFAT activation by pretreating HaCaTs with cyclosporin A. Induction of COX-2 by UVR is also wavelength-dependent and this is similar to the wavelength-dependence of NFAT. UVinduced apoptosis of HaCaTs is increased by inhibiting UV-induced NFAT activation. Taken together, these data implicate NFAT as being an important regulator of UV-induced COX-2 expression and apoptosis. The procarcinogenic and antiapoptotic role played by UVactivated COX-2 may be mediated, in part, by upstream NFAT signaling. It is also demonstrated that NFAT2 regulates keratinocyte proliferation. Overexpressing GFP/ NFAT2 increased proliferation of both HaCaTs and NHEKs and knockdown of NFAT2 using siRNA, reduced proliferation of HaCaTs. NFAT transcription factors may therefore play an important role in the pathology of hyperproliferative skin disorders and may be involved in UV-induced keratinocyte proliferation. UVR is involved in the pathogenesis of malignant melanoma (MM), the most aggressive form of skin cancer, which is notoriously unresponsive to current therapies. In this thesis, it is shown that the NFAT signaling pathway is functional in melanoma cell lines. NFAT transcriptional activity is inducible and cyclosporin-sensitive and both basal and inducible activity appears to correlate positively with the presence of a B-RAF mutation, shown to be present in up to 70% of MM. Melanoma cell proliferation is reduced by inhibiting NFAT with cyclosporin A, which induces an arrest in G1 phase of the cell cycle. COX-2 is a potential prognostic marker for MM and it is shown that COX-2 promoter activity and protein expression is regulated by NFAT. NFAT may be an important mediator of proliferation and invasion of melanoma via the induction of COX-2. Further studies are warranted to assess whether NFAT represents a novel therapeutic target for treatment of MM.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:485480 |
| Date | January 2008 |
| Creators | Flockhart, Ross James |
| Publisher | University of Newcastle upon Tyne |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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