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Title: Studies on the start family of lipid trafficking proteins in HaCaT keratinocytes
Author: Elbadawy, Hossein Mostafa
ISNI:       0000 0004 2722 0943
Awarding Body: Glasgow Caledonian University
Current Institution: Glasgow Caledonian University
Date of Award: 2011
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Terminally differentiating keratinocytes actively synthesize and accumulate lipids to maintain the production of lipid lamellae and an effective epidermal barrier. This thesis explored the role of steroidogenic acute regulatory (StAR) related-lipid transfer (START) proteins in the immortalized HaCaT keratinocyte cell line. Differing cell culture conditions were used to study differentiation of HaCaT keratinocytes, including the 'calcium switch' model, studied over a period of 21 days. Gene expression of five 'markers' of keratinocyte differentiation, KRTI, KRTIO, KRTI4, INVand LOR, was used to define progression of this process, revealing a dependency on calcium concentration, and increasing confluency. We then investigated gene expression of the START family of lipid trafficking proteins in HaCaT keratinocytes, compared with primary human keratinocytes. The same nine members of the family, STARDI, STARD2, STARD3, STARD4, STARD5, STARD7, STARDIO, STARD11 and STARDI2 were expressed in both cell types, although levels of STARD2, STARD5 and STARDI2 mRNA proved higher in HaCaT cells than in primary keratinocytes, and gene expression of STARD4 was lower in HaCaT cells compared with primary cells. During HaCaT differentiation, marked changes in gene expression of this family of proteins were noted, reflecting changes in lipid metabolism occurring during this process, the most notable being an increase in cholesterol mass after 21 days in cells exposed to I.4mM calcium compared with cells cultured in calcium depleted media. Steady state levels of mRNA encoding STARDI, D3, D2, D7, DID and Dll tended to increase during keratinocyte differentiation, while STARD4 and STARD5 showed a more complex pattern of expression, with decreased expression observed in the presence of I.4mM calcium. In order to investigate the function of the cytosolic cholesterol binding proteins, STARD4 and STARD5, in HaCaT cells, transient transfections were performed (48h) with pCMV6 vector encoding full length STARD4 or STARD5. Confocal microscopy confirmed the cytosolic location of both proteins, while in 3-D organotypic cultures STARD5 eo-localised with Keratin 10 rather than Loricrin, suggesting a locale in the spinous layer of these cultures. While both cholesterol trafficking proteins repressed cholesterol and cholesteryl ester biosynthesis from [I 4C] acetate, consistent with their proposed role as directional sterol transporters, these changes were associated with distinct gene expression patterns. Overexpression of ST ARD4 was associated with induction of Loricrin mRNA and protein, while STARD5 overexpression repressed gene expression of keratin 1, indicating that altered intracellular cholesterol transport can influence keratinocyte differentiation status. Further, ST ARD4 overexpression was associated with induction of SREBF2 and ABCG4, and repression of ABCAI, while overexpression of STARD5 resulted in induction of PPARD, PPARG and ABCAI, and repression of SREBF2 and LDLR. These findings confirm the impact of these cytosolic sterol transporters on keratinocyte lipid metabolism, and may suggest that ST ARD5 may be more efficient in this regard. In conclusion, manipulation of intracellular lipid transport proteins may therefore provide novel therapeutic strategies for the treatment of lipid-related skin disorders.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available