抄録
Background and aim: JNK (c-jun N-terminal kinase) is a key mediator of the inflammatory response and plays roles in multiple cellular processes in determining cell fates. Although regulatory pathway of JNK has been well documented to date, its dynamic behavior, as well as its regulation in living cells, remains elusive. We therefore aimed to elucidate JNK dynamics in living cells at higher resolution in time and space to understand its regulatory mechanism upon inflammatory cytokine stimulation.
Methods: HeLa cells were stimulated by IL-1β (10 ng/ml) and resulting JNK activity was quantified by FRET imaging.
Results: We developed a novel JNK reporter based on FRET and quantitatively evaluated JNK activity in HeLa cells. To elucidate regulatory mechanism of JNK signaling, we employed the systems-biology approach, by which the dynamic behavior of JNK signaling system can be examined based on the quantitative relationships between the "input" stimulation and the resulting "output" phenotype. We applied periodic pulsatile IL-1β stimuli as input then measured the stimulation frequency-dependence of resulting JNK activity (output). With this frequency-dependence of JNK activity, we found that the dynamics of cytokine-dependent JNK activity is governed by a slowly activated negative feedback regulation. We further demonstrated that the expression of a MAPK phosphatase, MKP-1, is involved in this negative regulation. Actually, the duration of JNK activity was restricted within about an hour even when IL-1β was given continuously to cells, and MKP-1 expression mirrored the JNK inactivation.
Conclusion: Our novel approach combining FRET cell imaging and systems analysis of signal transduction revealed that the dynamics and regulation of IL-1β dependent JNK activation are governed by a negative feedback regulation that involves the expression of a MAPK phosphatase, MKP-1. Such mechanism would be required to avoid excessive inflammatory response while properly maintaining the response to periodic cytokine stimuli.
