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Hormonal regulation and metabolic roles of CCAAT/enhancer-binding proteins



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The CCAAT/Enhancer-Binding proteins (C/EBPs) are liver-enriched transcription factors which are known to 'trans'-activate a number of metabolically important genes. The goal of this thesis work has been to advance areas of study on C/EBP isoform regulation and metabolic roles which have not been fully addressed in the current literature. The initial undertaking of this work involved the examination of the effects of hormones and diabetes on C/EBP isoform expression in rat H4IIE hepatoma cells and in rat liver. Treatment of cells with dexamethasone was observed to produce increases in C/EBPα and C/EBPβ mRNA and protein levels. Insulin was observed to produce an interesting bi-phasic response on C/EBPα expression. Treatment of H4IIE cells with 8-chlorophenylthio-cAMP produced greater inductive effects upon C/EBPβ expression than on C/EBPα expression. We observed an inhibition of C/EBPα gene expression in streptozotocin-diabetic rat liver which was reflected by decreases in both its mRNA and protein. However, an interesting alteration in the ratio of alternate C/EBPα translation forms was observed in the streptozotocin-diabetic livers suggesting a potential alteration in the 'trans'-activational activity of C/EBPα. These results suggest that hepatic C/EBP isoforms are under complex control by both hormonal and metabolic signals, which correlates well with their known role as 'trans'-activators of metabolically vital genes. Previous work has demonstrated a role for C/EBPα in mediating the cAMP responsiveness of synthetic phosphoenolpyruvate carboxykinase (PEPCK) promoter constructs within a transiently transfected cell culture system. In order to address the C/EBP isoform requirements for endogenous PEPCK gene expression and regulation, we have produced stable transfected hepatoma cells expressing antisense constructs for the two major C/EBP isoforms in liver. We demonstrate that targeted inhibition of C/EBPα but not C/EBPβ in rat hepatoma H4IIE cells significantly reduces the cAMP responsiveness of the endogenous PEPCK promoter. Cells expressing C/EBPα antisense were characterized by decreases in the levels C/EBPα mRNA and C/EBPα protein levels. The response of PEPCK to cAMP was marginal in C/EBPα antisense expressing cells, compared with a 3-fold induction of PEPCK expression by cAMP observed in wild-type H4IIE cells. The cAMP signaling pathway of C/EBPα antisense expressing cells was intact; in that the cAMP induction of the C/EBPβ gene was similar to that of normal H4IIE cells. Furthermore, the cAMP responsiveness of PEPCK in C/EBPβ antisense expressing cells was nearly identical to that of wild-type H4IIE cells. These data suggest that the α-isoform of C/EBP is specifically required for mediation of the cAMP response of endogenous PEPCK in rat hepatoma cells and cannot be functionally substituted for by C/EBPβ in this context.





Doctor of Philosophy (Ph.D.)







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