The long term objective of our research is to understand the relationship and the regulated expression of the inflammation-associated proteins Alpha1-MAP and the kininogens. In addition, the function of these proteins as antiinflammatory agents will be explored and their potential for clinical use investigated. The acute phase response to inflammation is a general reaction to tissue injury and is characterized by significant inductions in the levels of several serum proteins. Since the response is believed to facilitate recovery from a severe inflammation, studies of the proteins involved are applicable to numerous disease states. Many diseases including pulmonary emphysema, various forms of arthritis, nephrotoxic nephritis, and Arthus allergic reactions result from the damage to healthy tissue that occurs at an inflammatory site due to the excessive release of degradative enzymes from neutrophils in the area. The enzymes principally responsible for this destruction are collagenase, elastase, and a thiol-dependent protease. In rats, a cysteine proteinase inhibitor termed the major acute phase protein (Alpha1-MAP) is induced ten-fold in response to inflammation. Sequence analysis of a cDNA for Alpha1-MAP has demonstrated an unexpected homology to the kininogens which are the precursors of bradykinin and participate in the initiation of the clotting cascade.
The specific aims of our renewal grant are: (1) To isolate the gene or genes for the Alpha1-MAP acute phase protein of the rat including related genes such as the high and low molecular weight kininogens. This will be accomplished by screening rat genomic libraries using the Alpha1-MAP cDNA. (2) To characterize these genes with respect to exon-intron boundaries as well as determine the sequence of their 5' and 3' flanking regions. Standard mapping procedures, S1 nuclease analysis and DNA sequencing methods will be used in this effort. (3) To examine the tissue specific and developmental expression of these genes in rats. The presence of specific mRNAs before and after induction will be determined by a combination of S1 nuclease, primer extension and liquid hybridization analyses. (4) To investigate the regulation of the Alpha1-MAP gene in cell culture systems and transgenic mice. Intact genes, fusion genes and deletion and site specific mutants will be introduced into hepatoma cells by DNA mediated transformation and microinjected into mouse nuclei for studies in the resultant transgenic mice. (5) To study the role of Alpha1-MAP and kininogens in inflammation. This will be accomplished using an animal model for this response. (6) To determine if a homologous protein to Alpha1-MAP occurs in humans.
