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Title: Mechanisms of hyperkeratosis in autosomal recessive congenital ichthyosis (ARCI)
Author: Youssef, G.
ISNI:       0000 0004 8503 9982
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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Autosomal recessive congenital ichthyosis (ARCI) is an inherited skin condition, which clinically manifest as generalised epidermal scaling. The disorder arises largely from mutations in Transglutaminase 1 (TGM1) and Lipoxygenase 12b (ALOX12B). The aim of this project was to elucidate genetic signatures involved in hyperkeratosis in ARCI using Alox12b- and Tgm1-deficient cells (ARCI in vitro models). ARCI in vitro models were generated by transfecting rat epidermal keratinocytes (REKs) with shRNA against Alox12b mRNA and compared to pre-existing Tgm1 knock down cells (O'Shaughnessy et al., 2010). Array hybridisation analysis was used to compare the transcriptomic landscapes of Tgm1- deficient and Alox12b-deficient cells compared to controls. The list of differentially expressed genes was interrogated using supervised (analysis of the degree of enrichment of the differentially expressed ARCI gene list to signalling pathways) and unsupervised approaches (hierarchical clustering of differentially expressed genes). Tgm1- and Alox12bdeficient cells have distinct gene expression signatures with the former showing overrepresentation of genes implicated in fatty acid biosynthesis and the latter showing upregulation in genes associated with protein components of the epidermis. There were 139 commonly differentially expressed genes (ARCI hyperkeratotic signature) in the ARCI in vitro models which include keratin 1 and Akt3. Mdm2, TLR2 and Sumo 1 were identified in the scale-free network of functional interactions between the genes of the ARCI hyperkeratotic signature. The ARCI in vitro models were cultured in organotypic co-culture and exhibited hyperproliferation and thickened cornified envelope. Immunofluorescence and western blot analysis confirmed keratin 1, Mdm2, Akt3 and pAKT up-regulation as well as the up-regulation of TLR2/NF-kB pathway. Keratin 1 and Mdm2 up-regulation was also confirmed in ARCI skin biopsy samples. ARCI in vitro cells transiently transfected with Mdm2 shRNA or treated with the Mdm2 functional antagonist, nutlin-3, showed reduction in keratin 1 and pAkt levels. There were also reduced PCNA positive cells and a reduction in the thickness of the cornified envelope in nutlin-3 treated organotypic sections. ARCI in vitro models treated with the Tlr1/2 agonist, Pam3CSK exhibited elevated keratin 1, Mdm2, pAKT and Sumo 1 expression. These results indicate a novel mechanism for TLR2 signalling and Mdm2 in driving hyperkeratosis. We propose that TLR2 stimulation with an endogenous ligand results in Mdm2 up-regulation, which in turn stimulates pAkt to drive hyperproliferation and keratin 1 expression to contribute to the thickened cornified envelope.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available