Acute systemic injuries in the liver of adult mammals cause a drastic and coordinate increase in the production of a set of plasma proteins, the acute phase reactants. These proteins are essential in protecting the organisms against the harmful consequences of injury and for providing substrates for tissue repair. The overall goal of this project is to identify the potential effectors mediating this hepatic response, the interaction of the mediators with liver cells, and the intracellular events leading to the enhanced expression of acute phase plasma protein genes. It has been shown that activated monocytes and growing keratinocytes are major sources of structurally distinct factors, which when combined with glucocorticoids, will stimulate the synthesis of acute phase plasma proteins in cultured rodent and human liver cells as found in vivo. The following studies are proposed to elucidate the cellular and molecular mechanisms by which these factors control expression of two non-related and genetically unlinked plasma protein genes. 1) The cDNAs encoding the major hepatocyte-stimulating factors (HSFs) of human keratinocytes will be cloned. The deduced protein sequences will be compared to the principal liver regulating factors, interferon beta 2 and IL- 1 beta, of human monocytes. 2) The complexity and specificity of the plasma membrane receptors for these factors will be measured in liver cells, Hormone-receptor interactions will be correlated with the regulated expression of marker acute phase protein genes. HSF- and IFN-beta 2-specific receptors will be purified in order to assess potentially common functional properties. 3) The cis-located regulatory sequence through which HSFs, IFN-beta 2 and IL-1 modulate the activity of the rat alpha 1-acid glycoprotein and haptoglobin genes will be determined by testing the functionality of cloned and mutated gene fragments in transfected hepatoma cells. 4) The presence of specific nuclear or cellular proteins, recognizing compatable enhancer regions of the two genes, will be quantitated in cell-free binding assays. The function of the binding proteins as trans-acting factors will be verified by in vitro transcription analysis. And 5) the proteins with correct DNA-binding specificity and hormone-dependent activity will be purified. Immunologic and structural probes to the proteins will be generated and used to characterize the proteins' role in conveying the hormone receptor signal to the acute phase plasma protein genes.
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