. The goal of the proposed research is to elucidate the molecular mechanism of induction of serum amyloid A (SAA) protein synthesis in liver. SAA protein is an acute phase protein and is the precursor of amyloid protein A (AA), the main protein constituent of amyloid fibrils deposited in tissues in the disease associated with chronic inflammation e.g., rheumatoid arthritis, juvenile chronic arthritis. During periods of inflammation SAA protein synthesis in liver increases dramatically which has been found to be due to increased transcription of this gene. The objective is therefore to identify the regulatory elements of the SAA gene and the molecular events involved in this inducible biosynthetic process. To achieve these objectives investigators intend to specifically pursue the following sequences: (1) identification of DNA-binding domains that are required for the transcriptional activation; (2) determining the sequence of the regulatory regions; (3) characterization of these regions using a reporter chloramphenicol acetyl transferase (CAT) gene. Specific DNA binding domain, where transcription factor(s) bind to influence transcription of the gene, will be determined by DNA- protein gel retardation assay using fragments of the SAA genomic DNA. Identity and sequence of the binding domain will be determined by methylation interference assay. This regulatory structural element (s) will be characterized by ligating these sequences to an easily identifiable chloramphenicol acetyl transferase gene, whose transient expression in a transfected primary culture of hepatocytes will be followed in presence and in absence of SAA inducer (interleukin-1, interleukin-6). The potential importance of this project is to gain insight into cellular control mechanism of SAA gene induction. These studies will enable the formation of the basis for future work regarding the regulation of SAA biosynthesis in patients with disease which predisposes to amyloidosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15DK045144-01
Application #
3438043
Study Section
Metabolism Study Section (MET)
Project Start
1992-05-01
Project End
1994-04-30
Budget Start
1992-05-01
Budget End
1994-04-30
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Missouri-Columbia
Department
Type
Schools of Veterinary Medicine
DUNS #
112205955
City
Columbia
State
MO
Country
United States
Zip Code
65211
Ray, A; Hannink, M; Ray, B K (1995) Concerted participation of NF-kappa B and C/EBP heteromer in lipopolysaccharide induction of serum amyloid A gene expression in liver. J Biol Chem 270:7365-74
Ray, A; Ray, B K (1994) Serum amyloid A gene expression under acute-phase conditions involves participation of inducible C/EBP-beta and C/EBP-delta and their activation by phosphorylation. Mol Cell Biol 14:4324-32
Ray, B K; Ray, A (1994) Expression of the gene encoding alpha 1-acid glycoprotein in rabbit liver under acute-phase conditions involves induction and activation of beta and delta CCAAT-enhancer-binding proteins. Eur J Biochem 222:891-900
Ray, A; Ray, B K (1993) Analysis of the promoter element of the serum amyloid A gene and its interaction with constitutive and inducible nuclear factors from rabbit liver. Gene Expr 3:151-62
Ray, B K; Gao, X; Ray, A (1993) Regulation of rabbit alpha 1-acid glycoprotein gene expression in acute-phase liver. Identification of inducible and constitutive proteins like CCAAT-enhancer binding protein that interact with the 5'-proximal promoter elements. Eur J Biochem 216:127-36
Ray, B K; Ray, A (1993) Functional NF-kappa B element in rabbit serum amyloid A gene and its role in acute phase induction. Biochem Biophys Res Commun 193:1159-67
Ray, B K; Ray, A (1992) Identification of novel inducible nuclear factors that interact with the acute phase responsive promoter element of rabbit alpha 1-acid glycoprotein gene. Biochem Biophys Res Commun 189:1464-70