Characterisation of Claudin-1 Interaction with Ultraviolet Radiation in Human Epidermal Keratinocytes

Abstract

The skin forms the main barrier between the internal system of the body and the external environment. Skin barrier is composed mainly of two structures, the stratum corneum and tight junctions (TJs). Within TJs, Claudin-1 is a major barrier forming protein. Its absence in knock out mice results in death 24h after birth because of excessive dehydration. Although Claudin-1 is known to function within the TJs, unlike other claudins in the skin, Claudin-1 has a pan - epidermal expression pattern. Expression of Claudin-1 was previously observed to be reduced in basal layers after exposure of skin to Ultra Violet Radiation (UVR). In this study, Claudin-1 interactions with UVR were investigated with the aim being to characterise the mechanisms by which UVR affects Claudin-1 expression both within and outside of the TJ. UVR-Claudin-1 interactions were investigated by bioinformatics analysis followed by experimental work. Bioinformatics analyses show that Claudin-1, similar to the other claudins in the skin, has appreciable amount of UVR and Reactive Oxygen Species (ROS)- absorbing amino acids. In addition, bioinformatics predicts cleavage sites for UVR related proteases, namely matrix metalloproteases (MMP)-2, MMP-3, MMP-9 and Elastase-2. However, the bioinformatics analysis does not indicate characteristics for Claudin-1 which differentiate it from the other claudins. When investigated experimentally in undifferentiated keratinocytes, a single dose of Solar Simulated Radiation (SSR) caused significant loss of Claudin-1 72h post irradiation. The loss was blocked by a ROS scavenger and by MMPs inhibitor. In addition, zymography indicated activation of MMP-9 48h post SSR. Claudin-1 was also shown to be a substrate for both MMP-9 and MMP-3. It is speculated that ROS produced from SSR induced activation of MMP-9 which in turn caused proteolysis of Claudin-1 in undifferentiated keratinocytes. In differentiated keratinocytes, Claudin-1 expression was not affected by a single dose of SSR, but was delocalised to the cytoplasm along with two other major TJ proteins, Occludin and ZO-1. This was accompanied by loss of TJ function. A ROS scavenger protected the localisation of Claudin-1 but not the TJ function. Introducing an enzymatic anti-oxidant partially protected the function of the TJ. All these data suggest that the major mechanisms by which UVR affects Claudin-1 expression is via generation of ROS. In addition, these data could have implications for new therapies for UVR-related conditions where Claudin-1 is known to be lost such as in photoageing and the photodermatosis, Polymorphic Light Eruption.  

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Michael Sherratt (Supervisor) & Catherine O'Neill (Supervisor)