The effect of IKK2 inhibition on NF-kappaB activation and macrophage function in atherosclerosis

Abstract

Atherosclerosis is a chronic inflammatory disease, characterized by the accumulation of macrophage-derived foam cells in the vessel wall. One of the key regulators of inflammation is the transcription factor NF-kB, which controls the transcription of many genes with an established role in atherosclerosis. In resting cells, NF-kB is kept inactive by association with an inhibitory protein, IkB. NF-kB can be activated by a wide range of extracellular stimuli, resulting in activation of different signal transduction cascades, which eventually activate the IkB kinase (IKK) complex containing two catalytic subunits, IKK1 and IKK2 and a nonenzymatic regulatory component termed NEMO. IKK2 is the most important subunit for activation of the classical NF-kB pathway. Upon stimulation, the IKK complex phosphorylates IkB, inducing its ubiquitination and subsequent degradation. NF-kB is then free to translocate to the nucleus where it facilitates the transcription of many genes. In this study, we investigated the role of the transcription factor NF-kB in inflammatory signaling and lipid metabolism in macrophages by determining the effects of IKK2 inhibition in a monocyte/macrophage cell line on inflammation, apoptosis and foam cell formation, the three major processes involved in atherosclerosis. We used two methods to functionally inhibit IKK2. The first one was a retroviral approach to establish RAW 264.7 cells that stably express a dominant negative mutant of IKK2. A second method was the treatment of RAW 264.7 cells with the chemical IKK2 inhibitors SC-514 and SPC-839. This second method was used to validate the results obtained with the retroviral approach but also because these small molecules are very attractive agents for the use in future therapies. Our study provides some interesting observations. First of all, it was seen that the inhibition of IKK2 reduces the inflammatory response of macrophages. Second, the inhibition of IKK2 in macrophages increases the sensitivity of these cells to stimulus-induced cell death. Third, macrophages take up less modified LDL after functionally inhibiting IKK2, probably by down regulating the two main scavenger receptors, CD36 and SR-A, involved in lipid uptake. Overall, the effects reached with the retroviral approach were more pronounced than the ones induced with the chemical IKK2 inhibitors. This in vitro research can be important for future in vivo applications in atherosclerosis. Our finding that IKK2 inhibition reduces the inflammatory response and the uptake of lipids in macrophages is considered positive regarding atherosclerosis. The effect of IKK2 inhibition on apoptosis, on the other hand, can both be considered positive and negative. These data indicate that functionally inhibiting IKK2 might have opposing implications regarding atherosclerosis.