The goal of this project is to study the transduction signal pathways involved in the induction of CRP in liver cells. CRP is one of the acute phase proteins that shows a dramatic increase after cellular damage or inflammation. In tissue culture, IL-6 induces maximum CRP production and secretion in human hepatoma cells at a concentration of 500u/m1. Pretreatment of hepatoma cells with 1 nM TPA for 24 hr increases CRP production to about 5% of the I1-6 induced level, while higher doses of TPA abolish this stimulatory effect. Forskolin at 10-30 micromole M induces CRP production about four to eight times greater as compared to TPA. Incubation of liver cells with both TPA and Forskolin resulted in an additive effect of both drugs. These experiments indicate that part of the IL-6 induction pathway involves protein kinase A, and to a lesser extent protein kinase C. Since we found that the CRP promoter region contains two IL-6 responsive elements (IL-6RE), we constructed CAT expression plasmids with each of the elements independently. These plasmids were transfected into hepatoma cells, and the cells were treated with IL-6, TPA or Forskolin. We found that the distal IL-6RE responded almost exclusively to Forskolin but only at about 30-40% of the maximum IL-6 level with no additional effect when both TPA + Forskolin were combined. However, the proximal IL-6RE responded equally to both TPA an Forskolin and the combination of TAP + Forskolin resulted in an additive effect with an induction level about 40-50% greater than the maximum IL-6 activity.
