Herein, we dissected the role of Ron receptor TK in an endotoxin-induced model of acute liver failure to further studies demonstrating that mice with a global deletion of Ron TK had a protected liver injury phenotype (16
). Examination of hepatocytes and Kupffer cells showed expression of Ron in both, and importantly, ex vivo
experiments showed Ron signaling is critical for both suppressing hepatotoxic Kupffer cell cytokine production and for sensitizing hepatocytes to Kupffer cell-derived products such as TNFα. Together with in vivo
experiments using mice with either hepatocyte- or Kupffer cell-specific deletion of Ron TK, we determined that Ron TK signaling in both cell types has important effects on hepatocyte survival following exposure to endotoxin in this model of acute liver failure. However, the cell type likely responsible for Ron-dependent hepatic protection in this liver injury model is the hepatocyte, given that lack of Ron signaling in hepatocytes ex vivo
and in vivo
is sufficient to afford a hepatocyte survival benefit.
Following stimulation with LPS, TK−/− Kupffer cells have altered cytokine production ex vivo
compared to TK+/+ Kupffer cells, with the most significantly elevated cytokines being IL-1ra (2.5 fold), IL-6 (2 fold), TIMP-1 (1.5 fold), MCP-1 (3.5 fold) and MIP-2 (1.9 fold). A potential mechanism for these perturbations is increased NF-κB signaling in the Ron-deficient state. These perturbations in cytokine production in response to LPS are consistent with earlier in vivo
observations in this liver injury model (16
), as well as in models of endotoxic shock or nickel-induced acute lung injury (12
). Interestingly, the effect of the altered cytokine milieu generated by LPS-treated TK−/− Kupffer cells was more toxic to mouse hepatocytes in vitro
compared to TK+/+ Kupffer cells. This may be due to higher TNFα levels, the composition of cytokines, or the presence of an untested cytokine in the conditioned media. Moreover, it is possible that the kinetics of cytokine expression is as important as the composition of cytokines produced.
TK−/− hepatocytes are protected compared to TK+/+ hepatocytes from TNFα/ActD initiated cell death over the range of 0.5 to 5ng/ml of TNFα, which are similar to the serum TNFα levels observed in the previously reported in vivo
). Whether the observed differences in hepatocyte viability are sufficient to explain the in vivo
observations by Leonis et al.
is not discernable by this assay. Hepatocytes plated ex vivo
are without feedback mechanisms to Kupffer cells and other hepatic cell types, and thus, the magnitude of the effect of TNFα ex vivo
may not completely mimic what is observed in vivo
. However, the results from the conditional deletion of Ron selectively in hepatocytes support the significance of our ex vivo
culture conditions and prior in vivo
Previously, we demonstrated that the protected liver injury response to treatment with LPS/GalN in Ron TK−/− mice was associated with a 1–2 hour delay in the progression to death based on survival analyses (16
). The lack of a more significant discrepancy in the time to mortality may be multifactorial and confounded by the sensitivity of the Ron TK−/− mice to LPS alone and by the severe necrosis and endothelial damage that is observed in the model. Despite the modest overall survival benefit of the TK−/− mice compared to controls, less liver injury was observed in the TK−/− mice as judged by liver histopathology, ALT levels, hepatocyte TUNEL staining and the extent of hepatic apoptosis. Similar protected phenotypes were observed in the Alb-Cre Ron TKfl/fl
mice as the Ron TK−/− mice compared to control mice. This is based on a reduction in liver histopathology and significantly decreased ALT levels and TUNEL staining in the Alb-Cre Ron TKfl/fl
mice compared to controls. Interestingly, a 1- to 2-hour increase in survival time in the Alb-Cre Ron TKfl/fl
mice was also observed compared to controls. Therefore, the data is consistent with a protected liver phenotype in the Alb-Cre Ron TKfl/fl
mice compared to controls. Our data also show a significant decrease in survival of the LysCre mice and associated worsened liver phenotypes in these mice compared to Alb-Cre Ron TKfl/fl
and wild type mice and supports the premise that Ron functions in both cellular compartments in vivo
Our results show increased NF-κB activation in hepatocytes that lack Ron signaling. NF-κB inhibition has been shown to tilt the balance of TNFα signaling toward apoptosis in hepatocytes; the converse is found as well, that increased NF-κB signaling provides a survival benefit (6
). In addition, Ron has been shown to regulate NF-κB (12
). Pretreatment of primary hepatocytes with the NF-κB inhibitor Bay-11-7085 abrogated the survival advantage of Ron deficient cells, suggesting that the elevated NF-κB levels observed in the early time points in these cells may at least be partly responsible for the protective phenotype. Although the exact mechanism for how NF-κB activity protects cells from apoptosis is not clear, up-regulation of anti-apoptotic proteins is one important effect of NF-κB signaling. Several anti-apoptotic proteins can be up-regulated by TNFα through NF-κB, however, we have seen no difference in two such proteins, C-IAP-2 or XIAP (31
), between TK+/+ and TK−/− hepatocytes ex vivo
following exposure to TNFα when examined by western blotting (data not shown).
Thus, we have demonstrated that Ron signaling is detrimental to hepatocyte survival when challenged with TNFα and that Ron is a regulator of hepatotoxic cytokine signaling in Kupffer cells. While the exact mechanism has not been elucidated, our ex vivo and in vivo studies suggest that Ron signaling appears to limit NF-κB signaling in both hepatocytes and Kupffer cells, leading to an overall sensitization of hepatocytes to Kupffer cell-derived products. Further research on the cell-type specific effects of Ron, and on how Ron regulates NF-κB is important in order to understand the mechanisms underlying this receptor’s effects on hepatocyte survival and before positing strategies that may lead to therapies for ALF or other liver pathologies, such as obesity related steatohepatitis and alcohol induced liver disease, that may, in part, have liver injury mediated by endotoxin.