Ron receptor-dependent gene regulation in a mouse model of endotoxin-induced acute liver failure

Abstract

Background: Prior experimentation has shown that loss of the tyrosine kinase (TK) signaling domain of the Ron receptor leads to marked hepatocyte protection in a model of lipopolysaccharide-induced acute liver failure (ALF) in D-galactosamine (GalN)-sensitized mice. The aim of this study was to identify the role of Ron in the regulation of hepatic gene expression.

Methods: Microarray analyses were performed on liver RNA isolated sequentially from wild-type (WT) and TK-/- mice during the progression of ALF. Gene array data were validated using Western and immunohistochemistry analyses as well as with ex vivo culture systems.

Results: At baseline, 101 genes were differentially expressed between WT and TK-/- livers, which regulate processes involved in hypoxia, proliferation, apoptosis and metabolism. One hour after ALF induction, WT livers exhibited increased cytokine expression compared to TK-/- livers, and after 4 hours, an induction of suppressor of cytokine signaling (SOCS) genes as well as JAK-STAT pathway activation were prominent in TK-/- livers compared to controls.

Conclusion: Our studies suggest a novel hepato-protective mechanism in Ron TK-/- mice wherein increased and sustained SOCS production and JAK-STAT activation in the hepatocyte may inhibit the destructive proinflammatory milieu and promote survival factors which blunt hepatic death and the ensuing development of ALF.

Figure 1. 101 genes that are 1.5 fold changed in TK−/− livers compared to wild type (WT) livers basally

A) Gene Tree of all 101 dysregulated genes. B) Functional classification of a major subset of the genes based on biological process. Genes that were differentially expressed between untreated Ron TK−/− and WT mice were categorized using the DAVID GO term functional annotation tool. The number in parenthesis represents the total number of genes in those categories. C) Representative genes and their fold changes.

Figure 2. Gene expression changes in WT and TK−/− livers following 1 hour of endotoxin and GalN exposure

A) 312 genes whose expression was altered differently at 1hr after endotoxin exposure between WT and TK−/− livers. B) Cluster analysis of those 312 genes based on similarity of expression pattern. Utilizing a K-means algorithm from GeneSpring, six clustering groups were established to identify patterns of gene expression. The x-axis shows the genotypes and treatment times while the y-axis is the normalized intensity. C) 83 genes whose expression is altered similarly in both WT and TK−/− livers at 1 hr. D) Cluster analysis of 83 similarly regulated genes.

Figure 3. Suppressors of Cytokine Signaling are upregulated between 1 and 4 hrs in the TK−/− animals whereas in the WT animals their expression levels have returned to normal

The fold change of SOCS 1, 2 and 3 expression was examined at 0 to 1 hours and at 1 to 4 hours following LPS/GalN treatment. SOCS expression levels were maintained at high levels in the TK−/− livers compared to the livers from WT mice at the later time points suggesting an enhanced down regulation of cytokine signaling in the TK−/− animals compared to controls.

Figure 4. Increased expression and activation of JAK2 and STAT3 in TK−/− liver and hepatocytes

A) Western analysis depicting increased JAK2 protein levels in TK−/− livers 4 hours post LPS/GalN treatment. B) Densitometric quantification of Western blots for total JAK2 demonstrating a significant increase in JAK2 protein levels in TK−/− livers. C) Immunohistochemistry of liver sections from WT and TK−/− mice 4 hours post LPS/GalN treatment for STAT3 and JAK2 expression. Note the nuclear STAT3 localization in the TK−/− hepatocytes compared to controls. Scale bar = 50μm. D) Ex vivo treatment of WT and TK−/− hepatocytes with TNF-α and ActD demonstrates increased p-JAK2 and total JAK2 levels in the TK−/− hepatocytes compared to controls.

Figure 5. SOCS3 is upregulated in TK−/− Kupffer cells more than WT cells after LPS stimulation

Quantitative real time PCR analysis of LPS-stimulated Kupffer cells for the induction of SOCS3. TK−/− Kupffer cells displayed 1.8-fold more SOCS3 expression after LPS stimulation for 30 minutes compared to WT Kupffer cells.

Figure 6. Working model

Based on our data, a working model for the hepato-protective phenotype observed in Ron TK−/− livers of mice exposed to LPS/GalN was generated. We propose that high levels of SOCS genes during ALF seen in TK−/− liver antagonize the LPS-induced expression and function of inflammatory cytokines/chemokines in Kupffer cells as well as antagonize the deleterious effect of cytokine signaling in the TK−/− hepatocytes. High levels of SOCS, Bcl6 as well as higher NF-κB activity in TK−/− livers may also contribute to hepato-protection.