Tissue injury in mammals elicits an immediate defense reaction, known as the acute phase response. A network of interactive, cytokines and endocrine hormones is locally and systemically activated. The liver is one of the major target organs for these inflammatory factors and responds with a coordinate increase in the expression of several plasma protein genes. The acute phase plasma proteins in circulation are essential for neutralizing harmful consequences of the injury and re-establishing systemic homeostasis. The overall goal of this project is to delineate the regulatory mechanisms activated by the inflammatory cytokines in liver cells. Specifically, this proposal focuses on the molecular mode of action of IL-6 and IL-6-like cytokines. The studies address the interaction of the cytokines with plasma membrane receptors, the consequent activation of intracellular signalling reactions, the communication of the cytokine signal to the nucleus, and the transcriptional stimulation of the acute phase plasma protein genes. Cytokine action will be characterized by starting at the two ends of the signalling pathway: from the cytokine receptor that initiates signalling and from the genetic elements that serve as end target of the cytokine signal. The following aims have been set: 1) To define the DNA elements that mediate immediate transcriptional activation of the gene encoding rat beta-fibrinogen; 2) to identify in rat hepatoma cells the transcription factors that interact with these regulatory gene elements and to assess cytokine-induced changes in the concentration, structure, and function of these transcription factors; 3) to define the structural domains in the lL-6-type cytokine receptor subunits which are responsible for signal transduction; and 4) to characterize the interaction of intracellular proteins and kinases with the activated lL-6-type cytokine receptors. The functional dissection of the gene elements will involve transcription rate analyses of hybrid gene constructs that have been transiently introduced into cultured hepatoma cells. The cytokine effect on structure and function of specific nuclear proteins will be probed by immune precipitation, metabolic labelling and binding to identified regulatory gene sequences. The peptide segments within the cytoplasmic domain of gp130 and the receptors for leukemia inhibitory factor and granulocyte-colony stimulating factor that are necessary for signal transduction will be determined by expressing appropriately mutated receptors in transiently-transfected hepatoma cells and monitoring the subsequent stimulation of cytokine-responsive reporter genes. The cellular proteins specifically associated with the activated cyotokine receptor subunits will be recovered by either co- immunoprecipitation with receptor antibodies or by receptor affinity chromatography. The relationship of these proteins with protein kinases known to be involved in hormone signal communication will be established by biochemical and immunological comparisons. The functional relevance of the proteins for cytokine signal transduction will be assessed in hepatoma cell lines that are non-responsive to cytokines.
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