| dc.description.abstract | Keratin K2 is a 66 kDa type II intermediate filament protein expressed in differentiating keratinocytes of the epidermis with a very low-level expression in normal oral mucosa. In the epidermis it is expressed in the upper granular and spinous layers and is considered a marker of terminal differentiation. It is known to be upregulated in pre-cancerous lesions of the oral cavity. However, the mechanism of its induction in dysplasia and its role in oral cancer is not known. As it is only expressed in vivo, being down-regulated in cultured keratinocytes, there are no reported studies on the functions of this protein. Point mutations in this protein are associated with the skin condition Ichthyosis Bullosa of Siemens (IBS). Keratin filaments are in a constant state of assembly and disassembly to maintain cell stability and support. Keratins’ reorganisation and dynamics are affected mainly by their state of phosphorylation which explains the difference between health and disease and the molecular interactions between different keratin pairs as well as with other cytoplasmic proteins. Heat shock was shown to re-organises the keratin network which is mediated by phosphorylation. A model has been developed to study keratin filaments dynamics by introducing keratin K2, into a simple epithelial cell line, MCF-7 (breast carcinoma cell line) which lacks K2 expression but expresses other keratins including K8, K18 and K19. Introduction of K2 into MCF-7 cytoplasm allows it to bind and fully integrate into the pre-existing network. To understand the mechanism of K2 integration into the pre-existing filaments, the stability of the network and its phosphorylation state using two phosphatase inhibitors, Calyculin A (CL-A) and Okadaic Acid (OA) was studied. To investigate the response of keratin cytoskeleton to stress, 4 the effect of heat shock on filaments reorganisation was studided using immunocytochemistry and live cell imaging. The expression of K2 mRNA and protein was investigated in keratinocytes cell lines as well as in normal human epidermal keratinocyte (NHEK) along with other terminal differentiation keratins, K1 and K10. The absence of serum lipids (SLP) and phenol red (PR), which are generally used in culture medium, had a significant effect on the expression of these keratins at both mRNA and protein levels in NHEK. The effect was different compared to immortalised cell lines, which could be explained by immortalisation methods altering gene response. Adding back retinoic acid (ATRA) to the culture medium differentially affected the expression of these genes. Adding PR back into PR-free culture medium in NHEK did reduce the expression of K1 and K10 but not K2. To further investigate the effect of SLP, RA and PR, the post-transcriptional stability of K2, K10 and K1 mRNAs using Actinomycin D (AD) in NHEK cells was studied. Interestingly, K2 mRNA was stabilised whereas K1 and K10 mRNAs were destabilised by ATRA. These observations explain the differential effect of ATRA on the expression of these genes previously reported in the literature. Further investigations are required to decipher the mechanism(s) regulating transcriptional changes affected by different culture conditions and by RA. The aim of this project is to study keratin dynamics and the role of lipids on keratinocyte differentiation and mRNA stability. | en_US |
