Oval cell proliferation after chronic liver injury has been well documented in a variety of models. The association of oval cells with liver repopulation and carcinogenesis and their ability to differentiate bipotentially suggest that oval cells function as a liver progenitor cell compartment. Previous work has suggested roles for several cytokines in mediating the growth of oval cells 27282930313233
. However, a precise understanding of signaling pathways involved in oval cell proliferation is lacking. TNF-α has been shown to be a growth factor for an oval cell line in vitro 39
and has also been indirectly implicated in mediating oval cell proliferation in vivo 34
. In this study, we sought to determine whether TNF signaling is involved in the oval cell response in mice.
Using a modification of the CDE diet protocol for inducing oval cells in rats, we were able to induce oval cell proliferation in mice within 1 wk. Analysis of TNF-α mRNA levels showed that liver TNF rapidly increases after commencement of the diet and is further elevated during the initial stages of oval cell proliferation. Peak TNF levels were detected after 2 wk of CDE feeding. Expression of TNF in CDE liver was localized to oval and inflammatory cells; expression was not detected in hepatocytes. The early, sustained elevations in expression indicate that TNF may play a role in both induction and maintenance of oval cell proliferation in this model. In cultured oval cells, TNF reinitiates growth in arrested cells and induces proliferation through NF-κB activation 39
. It remains to be established whether NF-κB is a mediator of oval cell growth in vivo.
To directly evaluate the importance of TNF signaling in the oval cell response in vivo, we used KO mice lacking TNF R1 or TNF R2 and showed that signaling through the type 1 but not the type 2 receptor is essential for optimal oval cell response to the CDE diet. Compared with WT and TNF R2 KO animals, TNF R1 KO mice had fewer oval cells and a lower level of oval cell proliferation. The impairment of the oval cell response in TNF R1 KO mice was not a result of reduced damage caused by lack of TNF R1 signaling, because similar levels of fat accumulation, inflammatory cell infiltration, and serum ALT levels were observed in TNF R1 KO and WT mice. In addition, there was no difference between mortality of WT and TNF R1 KO mice on the diet in either the short or long term (data not shown), suggesting that the deficiency in oval cell proliferation did not impair liver regeneration. It is presumed that residual oval cell proliferation is sufficient to maintain parenchyma in the TNF R1 KO animals. Furthermore, although substantially impaired, liver regeneration is detected in the KO mice 36
. The CDE diet compromises the replicative capacity of hepatocytes. However, some hepatocyte growth may still occur, and even in the absence of TNF R1, hepatocyte regeneration may contribute to maintenance of liver function. Interestingly, an increase in the ratio of liver/body weight was observed in long-term TNF R1 KO mice. Untreated KO mice have been reported to have normal liver weight 37
, indicating that administration of the CDE diet, in the long term, has resulted in liver enlargement. The mechanisms underlying this finding have not been addressed but may involve proapoptotic signaling downstream of TNF R1. TNF R1 KO mice have impaired apoptosis in several models, including acute hepatic injury 5152
Current research suggests that TNF R1 is the dominant TNF receptor 5354
, and after partial hepatectomy, it is this receptor, not TNF R2, that is appears to be active in promoting hepatocyte growth 37
. The signaling pathway employed by regenerating hepatocytes proceeds via activation of NF-κB and IL-6 3644
. A similar mechanism is suggested for oval cell–associated liver regeneration, because dexamethasone, which inhibits oval cell proliferation 34
, inhibits NF-κB nuclear translocation 35
. However, divergence of the pathways mediating oval cell and hepatocyte regeneration must exist, as oval cell proliferation does not occur after partial hepatectomy. Evaluation of the oval cell response in IL-6 KO mice suggests that IL-6 is involved in oval cell proliferation induced by the CDE diet. However, while oval cell proliferation is impaired in IL-6 KO mice, the extent of impairment is lesser in these mice than in TNF R1 KO. This suggests that IL-6 is not the sole downstream factor responsible for the TNF R1–mediated effect. By contrast, after partial hepatectomy, IL-6 and TNF R1 KO mice both have profound deficiencies in hepatocyte replication 3644
. This indicates that the factors mediating oval cell proliferation induced by the CDE diet and hepatocyte replication after partial hepatectomy may differ with respect to involvement of IL-6, although both may require TNF R1 signaling. It is likely that the optimal oval cell response involves several other cytokines, which may include other members of the IL-6 family.
Oval cell proliferation is often seen in the early stages of disease states that lead to cancer in humans 55565758
and rodents 4859606162
. This implicates the oval cell compartment in the carcinogenic process. Based on this, it could be hypothesized that the number of oval cells would positively correlate with the likelihood of tumor formation. This could explain the inhibition of tumor development in TNF R1 KO mice. However, the possibility that TNF is itself mediating tumor progression cannot be excluded. TNF has been shown to direct differentiation of several cell types 636465
. Absence of TNF has also been shown to impair skin tumor formation 66
. TNF enhances growth factor mitogenicity 67
and may, therefore, affect transformation of these cells. Thus, while the experiments showed that absence of TNF R1 reduces tumor formation, the exact role of TNF signaling in the carcinogenic process is still unclear.
These studies, although they demonstrate a role for TNF signaling in oval cell proliferation, also highlight the complexity of the system mediating oval cell growth and differentiation. Absence of TNF R1 is sufficient to reduce oval cell numbers to approximately half of WT levels, but it does not completely abolish the oval cell response to CDE feeding. This indicates that additional growth factors or signaling pathways may supplement that of TNF R1 in oval cell signaling. Other cytokines may be induced for the different phases of the oval cell response: infiltration, growth, and differentiation. In this study, we have focused on the growth phase and show that TNF R1 is important for oval cell proliferation in this stage. By contrast, Matsusaka et al. 30
show that stem cell factor, while promoting liver oval cell infiltration, does not mediate the growth of these cells within the liver parenchyma. The association of a proinflammatory response with oval cell proliferation indicates that many cytokine systems may be activated during this process and that multiple factors may be required for the full oval cell response to liver damage. However, regardless of additional factors, this study demonstrates the involvement of TNF signaling in oval cell proliferation and carcinogenesis.