Transcriptional reprogramming in murine liver defines the physiologic consequences of biliary obstruction
Introduction
The timely activation of key physiologic processes is central to the reparative response of the liver to acute or chronic injuries. In chronic cholestasis, activation of non-parenchymal cells and progressive fibrosis dominate the response [1], while low-grade cellular proliferation is maintained to supply the critical cellular mass necessary to meet metabolic demands. In contrast, the response following an acute liver injury emphasizes cellular proliferation as the physiologic priority [2], [3]. The impaired hepatocyte proliferation and decreased survival following partial hepatectomy in mice lacking interleukin-6, tumor necrosis factor receptor, inducible nitric oxide synthase, and/or CCAAT enhancer-binding protein-β [4], [5], [6], [7] illustrate the genetic control of this response.
Similarly, cholestasis induces adaptive changes in hepatic gene expression, with an emphasis on maintaining intracellular homeostasis rather than restoring liver mass. For example, coordinate changes in the expression of genes encoding the sinusoidal sodium taurocholate cotransporter (NTCP), the canalicular organic cation transporter Mdr1, and cholesterol 7-α-hydroxylase minimize the accumulation of toxic bile acids within hepatocytes by decreasing both uptake and de novo synthesis [8], [9], [10]. In order to more broadly define the consequences of cholestasis on liver gene expression, we carried out a large-scale analysis of hepatic gene expression following bile duct ligation (BDL). We hypothesized that biliary obstruction results in transcriptional reprogramming that dictates the physiologic response. We found that following biliary obstruction there is a time-restricted and sequential pattern of gene activation, beginning with induction of cholesterol metabolism, followed by activation of cellular proliferation, and finally by production and remodeling of the structural matrix.
Section snippets
Bile duct ligation
BDL was performed in 4–6 week-old female mice of mixed genetic background (C57Bl/6-SJL-Swiss Black). Using anesthesia with a 2% isofluorane/98% room air mixture, an abdominal incision was followed by retraction of the duodenum and dissection of the bile duct toward the hepatic hilum, where it was completely transected between two sutures. The abdominal wall was then sutured, and the mouse was allowed to awake from anesthesia. To demonstrate complete occlusion of the biliary system, we injected
Reprogramming of liver gene expression following biliary obstruction
Gene expression analysis revealed 92 transcripts expressed ≥2-fold above controls at one or more time points following BDL. Grouping of these genes based on level of expression and time after BDL identified two distinct patterns (Fig. 1A; Table 2). The first was a unique overexpression of 27% of genes at specific time points following BDL, with baseline levels of expression at all other time points. In the second, 73% of transcripts were highly sensitive to biliary obstruction and displayed
Discussion
Our large-scale analysis of liver gene expression following biliary obstruction demonstrates an organized pattern of gene activation that reflects the timely initiation of specific physiologic processes in response to impaired bile flow. Although there was a dominant activation of metabolic genes at all phases, involvement of specific pathways varied according to the duration of obstruction. Overall, the reprogramming in gene expression occurred in a time-restricted and sequential fashion
Acknowledgements
The authors thank Bruce Aronow for tutorial in use of gene expression software and preliminary analysis of gene expression profiles, and Drs Dong-Yi Zhang and Pranavkumar Shivakumar for technical assistance and support for real-time PCR. We also thank Drs William F. Balistreri, John C. Bucuvalas, and James E. Heubi for insightful review of the manuscript. This work was supported in part by the NIH- R01-DK064008 grant and the Translational Research Initiative of Cincinnati Children's Hospital
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