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Title: Studies into the regulation of CCAAT Enhancer Binding Protein delta expression (C EBP) delta by cytokines
Author: Ali, Saira
ISNI:       0000 0004 2750 3046
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2007
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Inflammation is a physiological response to injury, trauma or infection. The initial phase of inflammation, the acute phase response (APR), is characterised by changes in levels of serum acute phase response proteins (APPs). These APPs are synthesised primarily by hepatocytes in response to several inflammatory cytokines, including interleukin-1 (IL-1) and interleukin- 6 (IL-6). Expression of APP genes is known to be regulated by the CCAAT Enhancer Binding Protein (C/EBP) family of transcription factors, which consists of six members (a-Q. Several studies have implicated C/EBP5 as an important regulator of APP gene transcription during inflammation. Additionally, a number of cytokines and other inflammatory mediators modulate the expression/activity of C/EBP8 during the APR. Unfortunately, the mechanisms by which cytokines regulate the expression of C/EBP5 remain poorly understood. Understanding these mechanisms could potentially aid in the development of therapeutic strategies aimed at combating inflammatory disease. Therefore the objective of this project was to delineate the molecular mechanisms governing C/EBP5 expression in response to IL-1 and to a lesser extent, IL-6. Initial investigations were designed to determine the effects of various inflammatory mediators on C/EBP5 expression in hepatocytes focusing on IL-1, with the aim of delineating the regulatory elements in the human C/EBP5 gene promoter that were responsible for gene expression in response to this cytokine, and to identify the nuclear factors regulating this response. However, unlike endogenous C/EBP5 mRNA and protein expression, both of which were induced by IL-1, the activity of the human C/EBP5 gene promoter was not stimulated by this cytokine, despite being responsive to IL-6 action. Similar results were also obtained when the actions of IL-1 and IL-6 on the murine C/EBP5 gene promoter were examined. Using the same transfection system, studies were also initiated to determine if the human C/EBP6 gene was subject to auto-regulation, as this mode of control is likely to play a significant role in regulating C/EBP5 gene expression during the APR and because this was a novel area of investigation. Results from these studies showed, for the first time, that the human C/EBP8 gene is subject to auto-regulation, potentially via an indirect mechanism and the cw-acting elements responsible for this response were identified to within approximately 200 base pairs of the transcriptional initiation site. Therefore, as a consequence of our initial findings, we adopted an alternative strategy for determining the mechanisms by which C/EBP5 expression was induced by IL-1. This involved investigating the signal transduction pathways involved in the actions of IL-1 on the expression of this gene and characterising the critical components of these pathways. Simultaneously, some investigations were also carried out to identify the transcription factors regulating endogenous C/EBP5 gene expression by IL-6, given that this was also a novel area of investigation. With the use of a range of commercially available pharmacological inhibitors, our initial investigations led to the identification of casein kinase 2 (CK2), c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-kB) as potential regulators of IL-1-induced C/EBP6 expression. The exact role of these pathways was then further investigated using several independent approaches. Western blot analysis and associated kinase activation assays revealed that JNK is activated by IL-1 in hepatocytes, a novel finding. With the use of RNA interference, /rum-activation assays and electrophoretic mobility shift assays, our investigations led to the identification of NF-kB as a key regulator of the IL-1-induced C/EBP6 expression, also a novel finding. In association with this finding, we also investigated a role for CK2 and JNK in the regulation of NF-kB activation by IL-1. Finally, our studies have also revealed a critical role for the STAT (Signal Transducer and Activator of Transcription) family of transcription factors in the regulation of human C/EBP6 gene expression by IL-6. Consistent with this finding, we also show that IL-6 action leads to the activation of both STAT1 and STAT3 in hepatocytes. To summarise, the studies presented here have revealed that the human C/EBP8 gene is subject to transcriptional regulation by auto-activation and in response to IL-1 and IL-6 action is induced by NF-kB and STATs, respectively. These studies have provided novel insights into the molecular mechanisms by which C/EBP5 is regulated during the APR.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available