Abstract
Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.
Highlights
Wnt3a secretion from macrophages maintains the expression of the Notch inhibitor, Numb, in hepatic progenitor cell (HPC) promoting their differentiation to hepatocytes[9]
We have developed an in vivo platform for quantifying hepatocyte transcription factor activity in live mice
The role of TGF-βsignaling is well characterised in hepatic stellate cell (HSC), activity in hepatocytes after liver injury is less clear
Summary
Wnt3a secretion from macrophages maintains the expression of the Notch inhibitor, Numb, in HPCs promoting their differentiation to hepatocytes[9]. We used lentiviral transcription factor activated reporter cassettes (TFAR) co-expressing firefly luciferase and eGFP. Conditional control of reporter gene activity by a synthetic promoter enabled longitudinal analysis of transcription factor activity profiles by continued bioimaging in mice following pBDL. Intravascular injection of VSV-G pseudotyped lentiviruses into newborn mice resulted in almost exclusive transduction of hepatocytes, which allowed analysis of hepatocyte-specific transcription factor activity. This has enabled us to longitudinally assess hepatocyte NF-κB, Smad 2/3, Notch and Wnt signaling activity in vivo following pBDL. We combined TFAR bioimaging with complementary immunohistochemistry and in vitro evaluation of hepatic progenitor cell (HepaRG cells) differentiation to provide new insights into the temporal transcriptional activity and signaling mechanisms that mediate the hepatic injury and repair response during cholestatic liver injury
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