Mechanisms of nuclear factor kappa B (NF-κB) activation in response to LPS and hypoxia

Cytokines such as tumor necrosis factor alpha (TNFalpha) are signaling elements involved in the pathophysiology of the systemic inflammatory response in critically ill patients with sepsis. During systemic sepsis or in response to experimental lipopolysaccharide (LPS) challenge, the uncontrolled release of TNF-alpha into the circulation contributes to the circulatory dysfunction and may precipitate the development of organ injury in multiple organs including the lungs. Lung gas exchange failure and circulatory disturbances can lead to tissue hypoxia, which can damage tissues by itself. The transcription factor nuclear factor kappa B (NF-kappaB) stimulates the transcription of pro-inflammatory cytokines including tumor necrosis factor alpha (TNFalpha). LPS and hypoxia both induce NF-kappaB activation and TNFalpha gene transcription. Furthermore, hypoxia augments LPS induction of TNFalpha mRNA. Hypoxia stimulates NF-kappaB and TNFalpha gene transcription and increases ROS generation as measured by the oxidant sensitive dye 2', 7'-dichlorofluorescin-diacetate (DCFH-DA). The antioxidants N-acetylcysteine (NAC) and pyrollidine dithiocarbamic acid (PDTC) abolished the hypoxic activation of NF-kappaB, TNFalpha gene transcription, and increases in ROS levels. Rotenone, an inhibitor of mitochondrial complex I, abolished the increase in ROS signal, the activation of NF-kappaB, and TNFalpha gene transcription during hypoxia. LPS stimulated NF-kappaB and TNFalpha gene transcription but not ROS generation. Rotenone, PDTC, and NAC had no effect on the LPS stimulation of NF-kappaB and TNFalpha gene transcription, indicating that LPS activates NF-kappaB and TNFalpha gene transcription through a ROS-independent mechanism. These results indicate that mitochondrial ROS are required for the hypoxic activation of NF-kappaB and TNFalpha gene transcription, but not for the LPS activation of NF-kappaB.