Cellular response to inflammatory cytokines involves concerted changes in gene expression. Cytokines, such as IL-6 and TNF-α, regulate gene expression through multiple intracellular signaling pathways. The activation of transcription factors is one of the important mechanisms through which these cytokines regulate gene expression, and NF-κB is a key transcription factor that is activated by TNF-α during inflammation. In this study, we have utilized green fluorescent protein reporter cells along with fluorescence microscopy, image analysis and mechanistic modeling to determine the activation dynamics of NF-κB in H35 rat hepatoma cells upon TNF-α stimulation. NF-κB reporter cells were monitored for induction of GFP expression for 24 hours following continuous stimulation with 2.5ng/mL, 10ng/mL and 25ng/mL TNF-α. As expected, TNF-α addition resulted in a significant increase in fluorescence. Relative fluorescence profiles were generated from the fluorescence intensity data, and indicated that fluorescence increases up to 24 hours after an initial delay of approximately four hours. The fluorescence data was also used to develop a model describing significant events leading to NF-κB activation and GFP expression. In addition, a model describing regulatable expression of GFP upon stable integration into the genome was also developed. Comparing these two models led to the construction of a third model depicting NF-κB activation dynamics. Simulation of the model representing NF-κB activation dynamics yielded an NF- κB activation profile, which demonstrated that in the presence of constant TNF-α stimulation, there is an approximate 90 minute hour time delay followed by a rapid increase in nuclear NF-κB, that reaches a steady state value at approximately two hours. This study establishes a method to derive NF-κB activation from reporter cell fluorescence data, and can be used to infer dynamics of activation of other transcription factors using GFP reporter cell lines.
| Identifer | oai:union.ndltd.org:TEXASAandM/oai:repository.tamu.edu:1969.1/ETD-TAMU-1443 |
| Date | 15 May 2009 |
| Creators | Laible, Allyson Marie |
| Contributors | Jayaraman, Arul, Hahn, Juergen, McShane, Mike |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | thesis, text |
| Format | electronic, application/pdf, born digital |
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