Serum amyloid A (SAA) is a prominant acute phase reactant in humans and precursor to the subunit protein comprising reactive amyloid deposits. In response to tissue injury the serum concentration of SAA rises as much as 500-fold. This increase reflects a cytokine-mediated induction of SAA synthesis by the liver. The purpose of this tremendous increase has not been determined. However, sustained high levels of SAA due to chronic inflammatory conditions or recurrent infections are known to predispose to reactive amyloidosis. Two major isotypes of SAA are present in human serum. Results from this laboratory indicate that the concentration of SAA1 is significantly higher than that not exclusive, form in amyloid deposits. Based on these observations, this study is designed to define the mechanisms which regulate the differential expression of human SAA isotypes following induction of the acute phase. Potential sites of regulation at the levels of SAA genes, mRNA, and protein will be evaluated in 4 specific aims: 1) Relative levels of SAA1 and SAA2 in the serum of patients who have either an isolated acute phase response, chronic inflammation, or reactive amyloidosis will be determined by quantitative electrofocusing. 2) Transcriptional activity of SAA1 and SAA2 genes will be compared. As a prerequisite to this aim, regulatory and structural regions of the two genes will be cloned and sequenced. Promoter and enhancer elements will be evaluated by measuring transcript levels generated from a series of SAA promoter-minigene constructs. 3) Steady state levels of SAA1 and SAA2 mRNAs will be compared by northern analysis and cell-free translation assays. Half-lives of the mRNAs also will be evaluated. 4) SAA1 and SAA2 proteins will be compared in terms of in vitro affinities for HDL and in vivo plasma clearance rates. Both experiments will utilize biosynthetically radiolabeled recombinant SAA proteins. The fact that SAA1 and SAA2 are present in serum at different levels suggests that the biologic properties and activities of the two isotypes may also differ. Defining the mechanisms by which these levels are regulated may shed light on yet undetermined functions of SAA. Furthermore, accomplishing these aims may provide clues essential to understanding the pathogenesis of amyloid formation and, in particular, the preferential deposition of SAA1.
