Following partial hepatectomy, a coordinated series of molecular events occurs to regulate hepatocyte entry into the cell cycle to recover lost mass. In rats during the first six hours following resection, hepatocytes are primed by a tightly controlled cytokine response to prepare hepatocytes to begin replication. Although it appears to be a critical element driving regeneration, the cytokine response to resection has not yet been fully characterized. Specifically, the role of one of the key response elements to cytokine signaling (NF-κB) remains incompletely characterized. In this study, we present a novel, genome-wide, pattern-based analysis characterizing NF-κB binding during the priming phase of liver regeneration. We interrogated the dynamic regulation of priming by NF-κB through categorizing NF-κB binding in different temporal profiles: immediate sustained response, early transient response, and delayed response to partial hepatectomy. We then identified functional regulation of NF-κB binding by relating the temporal response profile to differential gene expression. We found that NF-κB bound genes govern negative regulation of cell growth and inflammatory response immediately following hepatectomy. NF-κB also transiently regulates genes responsible for lipid biosynthesis and transport as well as induction of apoptosis following hepatectomy. By the end of the priming phase, NF-κB regulation of genes involved in inflammatory response, negative regulation of cell death, and extracellular structure organization became prominent. These results suggest that the immediate, transient, and delayed NF-κB signaling serve different functional transitions that drive the onset of regeneration.
Overall design: Adult (8-10 week old) Sprague-Dawley rats were given ad-libitum access to food (Chow) and water. When their weight reached 275-350 g, they were anesthetized and subjected to 70% partial hepatecomy by surgical removal of medial and left lateral lobes as per standard procedure. The medial and left lateral lobes were flash frozen using liquid nitrogen-cooled aluminum clamps to serve as within-animal, 0 hour controls. At 1 hour, 2 hours, 4 hours, and 6 hours post-PHx, rats (three biological replicates per time) were again anesthetized and the remnant liver tissue was excised and flash-frozen as before. Following excision of the remaining liver mass, rats were sacrificed by cervical dislocation. Liver tissue from 0h, 1h, 2h, 4h, and 6h post-PHx was subjected to chromatin immunoprecipitation. Immunoprecipitated samples (ChIP) from 0h, 1h, and 6h post-PHx were used to identifiy genome-wide NF-κB binding sites using microarrays (ChIP-chip). Immunoprecipitated samples from 0h, 1h, 2h, 4h, and 6h post-PHx were used for validation and extension of ChIP-chip results using qPCR.
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