SAA IS A NOVEL ACTIVATOR OF NF-kB-DEPENDENT GENE EXPRESSION IN INTESTINAL EPITHELIAL CELLS

HB Jijon, KL Madsen, C Jobin

BACKGROUND: Serum Amyloid A is an acute phase protein synthesized in the liver and the intestine whose levels increase up to 1000 fold during inflammation and correlate with disease activity in Crohn’s disease. Recent studies have shown SAA to activate NF-kB in leukocytes. We hypothesized that SAA activates NF-kB and modulates pro-inflammatory gene expression in intestinal epithelial cells (IECs).

METHODS: HT-29 and Caco-2 IECs were stimulated with 10 ng/ml TNF-a, 5 ng/ml IL-1 or 2-10 uM SAA and NF-kB activity measured as an index of epithelial pro-inflammatory gene expression. NF-kB activity was determined by EMSA, reporter assays, ChIP, gene expression (RT-PCR, ELISA) and IkB degradation, following stimulation in the presence or absence of adenoviral vectors encoding dominant negative mutants to NF-kB signaling proteins. Western blots against the active forms of various kinases were also performed. SAA treatment of IECs resulted in increased IL-8 and COX-2 mRNA expression, IL-8 protein secretion, IkB degradation, enhanced kb-luciferase expression, and NF-kB DNA-binding. Infection of cells with dnIKKb or non-degradable IkB abrogated IL-8 mRNA synthesis and IL-8 secretion. Likewise, co-infection with an adenoviral vector encoding kb-driven luciferase, and dominant negative IKKb or IkB resulted in a marked reduction in reporter activity. In addition, SAA stimulated MAPK activation, AKT activation and p65 (S536) phosphorylation. Furthermore, SAA augmented IL-8 secretion in response to IL-1 and TNF-a. Interestingly, although SAA has been suggested to signal through the G_i-coupled, Ca⁺⁺-dependent FPRL receptor, SAA-dependent IL-8 mRNA expression was not sensitive to PTX or BAPTA.

CONCLUSIONS: SAA activates and potentiates NF-kB activation in IECs through the classical IKK/IkB pathway in a Ca⁺⁺ and G_i-receptor independent manner, and activates multiple MAPK pathways. SAA may play an important role during intestinal inflammation.