STRUCTURE AND REGULATION OF LIVER ACUTE PHASE GENES

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This proposal is designed to test the hypothesis that the dramatic increase in serum concentrations of rat alpha2-macroglobulin (A2M) and rabbit C-reactive protein (CRP), which occurs in acute inflammation, is due to a corresponding increase in transcription from the respective genes. The goal is to characterize a system to study the inflammatory gene regulation in cultured liver cells and to identify the cis-acting control sequences of these genes. The project comprises five stages: 1) Preparation of a panel of rat and rabbit liver cDNA clones to be used as nucleic acid hybridization probes. The panel includes rat A2M and complement components C3 and C5, which are structurally related but are not major acute phase proteins. 2) Isolation and partial characterization of the A2M and CRP genes as source of control region DNA for use in transformation experiments. Sequence analysis of the rat A2M gene. 3) Titration of the corresponding liver RNA concentrations during experimental inflammations. Attempt to discriminate between transcriptional and post-transcriptional control mechanisms by measuring rates of transcription in liver nuclei from stimulated and non-stimulated animals. Visualization of recruitment of new cells for transcription by in-situ hybridization of liver sections. 4) Development of short-term and long-term culture conditions under which rat and rabbit hepatocytes can express acute phase mRNAs. An effort will be made to define conditions of treatment of the cultures with serum factors from stimulated animals, in order to achieve increased transcription of acute phase genes. For long-term cultures an established rat hepatocyte cell line will be used which is transformed with a temperature sensitive (tsA) mutant of the DNA-tumor-virus SV40. Expression of liver-specific genes can be induced in these cells by a shift in the culture temperature from 33 degrees C to 40 degrees C. 5) Identification of cis-acting control sequences of the A2M and CRP genes involved in the inflammatory regulation. Construction of test plasmids carrying control sequences from an acute phase gene and an assayable marker. Packaging of these DNA sequences into adenovirus 5 particles. Infection of liver cell cultures, induction with inflammatory stimuli and assay for increased transcription originating from the control sequences of the acute phase genes. Dissection of the control sequences by recombinant DNA manipulations (deletions, linker insertions, fragment exchange).