Adhesion molecule gene expression in response to inflammation: The role of NF-kappaB
Description
The vascular endothelium, by virtue of its location, is first to receive the effects of injury resulting from ischemia/reperfusion, Adhesion molecules play a role in the pathogenesis of myocardial ischemia and reperfusion injury. In endothelial cells, cytokines such as TNF$\alpha$ cause an increase in the gene expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Studies were done to determine if changes in oxygen levels have an effect on the expression of ICAM-1 in human coronary artery endothelial cells (HCAECs). Differential display was used to screen populations of mRNA isolated from HCAECs exposed to hypoxia. This study provided preliminary data showing that HCAECs are resistant to periods of hypoxia or anoxia and that ICAM-1 PCR-band intensity increases after 12 hours of hypoxia followed by 4 hours of reoxygenation. Furthermore, several potential candidates for increased hypoxia-mediated gene expression were isolated by differential display. A second study explored the role NF-$\kappa$B in adhesion molecule gene expression in response to cytokines such as TNF$\alpha.$ Nuclear activity of NF-$\kappa$B is regulated by the retention of NF-$\kappa$B in the cytoplasm by the inhibitor protein I$\kappa$B. I$\kappa$B mutations consisting of a deletion of the 36 N-terminal amino acids (I$\kappa$B $\Delta$N) expressed in human umbilical vein endothelial cells (HUVECs) were made using a retroviral transduction system. Steady state mRNA expression of both ICAM-1 and E-selectin was increased in response to TNF$\alpha$ after 4 hours. Also, NF-$\kappa$B binding activity was increased after 30 minutes of TNF$\alpha$ treatment. Furthermore, a preliminary experiment showed that overexpression of I$\kappa$B $\Delta$N resulted in decreased expression of a luciferase gene driven by an NF-$\kappa$B containing section of the E-selectin promoter in response to TNF$\alpha.$ Examination of HUVECs expressing I$\kappa$B N-mutations by Western blot demonstrated that I$\kappa$B $\Delta$N is resistant to degradation and remains complexed to NF-$\kappa$B p65 after treatment with TNF$\alpha.$ Furthermore, examination of nuclear extracts made from HUVECs treated for 4 hours showed that in those cells expressing I$\kappa$B $\Delta$N, NF-$\kappa$B activity was undetectable and NF-$\kappa$B modulated adhesion molecule expression was inhibited