This report shows that TWEAK selectively stimulates proliferation of a hepatic progenitor cell population known as oval cells both when overexpressed in transgenic mice and when transiently expressed in adult mice. This mitogenic effect of TWEAK is mediated exclusively through Fn14. In addition, the physiological role of TWEAK as a novel stimulator of oval cell proliferation is demonstrated in the DDC liver injury model, which features progenitor cell expansion. Since existing models of oval cell proliferation appear to require blockade of hepatocellular proliferation, we examined TWEAK-Tg mice for the presence of this condition and found no evidence for hepatic damage. In addition, we found that TWEAK pathway blockade had no effect on the regenerative response to PH (A. Jakubowski and D.M. Bissell, unpublished observations). Previous studies have provided evidence that HGF, TGF-α, EGF, FGF, TNF-α, IL-6, and leukemia–inhibitory growth factor (LIF) modulate oval cell proliferation in the context of injury (34
). However, these growth factors also modulate hepatocyte proliferation in response to injury (10
). In contrast, TWEAK appears to be selective for oval cells, with no detectable mitogenic effect on hepatocytes, and represents the first such factor described.
The hyperplastic phenotype observed in the livers of both TWEAK-Tg mice and Ad-TWEAK–treated mice resembles that found in mice fed with choline-deficient, ethionine-containing (CDE) diet as well as in the AAF/PH model of oval cell proliferation (7
). In the CDE model, individual OV-6– and α-fetoprotein–positive periportal oval cells appear early, then differentiate into duct-like structures and later into hepatocytes (40
). Similarly, individual unorganized oval cells were observed in 2-week-old TWEAK-Tg mice; these preceded the appearance of duct-like structures, found in adult 8-week-old mice, which suggests that these cells represent a less mature differentiation stage. As previously reported, progenitor cells expressed both A6 and CD34 markers (25
). Some progenitor cells expressed CD34 but not A6, which suggests that they represent a less mature population (26
). Thus, it is likely that the observed oval cells represent various maturation stages, up to definitive BECs. However, A6 and CD34 markers are also expressed on mature BECs, and therefore we cannot rule out the possibility that some of the proliferating cells were BECs. Whether or not the TWEAK-elicited cells mature into hepatocytes, as do carcinogen-induced oval cells (7
), will be the focus of a future study. It will be important to determine whether TWEAK acts solely to expand the progenitor population or is also a differentiating factor for the biliary pathway. Interestingly, we observed no progression of the hyperplastic phenotype leading to oval cell accumulation in older animals nor increased liver mass. Instead, oval and ductal cells, but not hepatocytes, had a higher turnover in TWEAK-Tg mice compared with control mice, suggesting that TWEAK may contribute mainly to initiating progenitor cell expansion.
Fn14 is expressed on the ductal plate in the embryonic liver and on the biliary duct epithelium and a few periductal cells in the adult normal liver; such cells may include the hepatic stem cells that presumably reside in or near the canals of Hering. These findings further support the conclusion that TWEAK directly delivers growth signals to progenitor cells and BECs in vivo. It has been reported that hepatic stellate cells proliferate in close proximity to oval cells, elaborating growth factors that are mitogenic for oval cells (43
). The participation of stellate cells in the induction of the hyperplastic phenotype cannot be excluded since they may also express Fn14 (D.M. Bissell, unpublished observations). However, this is less likely since the number of periportal cells with smooth muscle actin was unchanged in TWEAK-Tg mice, indicating an absence of activated stellate cells (data not shown). The role of TWEAK in driving oval cell proliferation was demonstrated also in the DDC chemical injury model for oval cell expansion, in which blocking anti-TWEAK mAb resulted in 35% inhibition of oval cell proliferation. A similar level of inhibition was also obtained in the DDC-fed Fn14-KO mice, thus supporting the conclusions obtained with the pharmacological inhibition: that the TWEAK/Fn14 pathway plays a role in stimulating oval cell proliferation. Since TWEAK blockade as well as the Fn14 deficiency did not result in complete inhibition of oval cell proliferation, other pathways appear to be involved in this function. We found that Fn14 mRNA was upregulated in oval cells and BECs in the reactive portal area of DDC-fed mice. The source of endogenous TWEAK in this model is speculative, but most likely the source is Kupffer cells or infiltrating macrophages, as suggested by TWEAK mRNA expression in monocyte/macrophage cell populations (17
). Although Fn14 was also weakly expressed on scattered hepatocytes, we found no evidence that TWEAK produced in the portal zone has any effect on lobular hepatocytes.
Recently, Polek et al. (44
) examined the differentiating effect of TWEAK on a murine monocyte/macrophage cell line that does not display Fn14. Under the influence of TWEAK, the cells acquired the features of osteoclasts, which suggests the possibility of an additional TWEAK receptor. The nature of this putative receptor and its tissue distribution remain to be determined. In the liver, the mitogenic effect of TWEAK appears to be mediated solely by the known TWEAK receptor, Fn14.
The classical models of oval cell proliferation involve administration of carcinogens (7
). In general, these compounds block hepatocyte proliferation, and studies have shown that neoplasia in these models does not arise by dedifferentiation of mature hepatocytes. Thus, it is thought that cancer arises out of transformed oval cells (3
). Also, oval cells have been identified in human liver diseases associated with a high incidence of HCC or cholangiocarcinoma (such as chronic viral and alcoholic hepatitis, NASH, and sclerosing cholangitis) (43
). Our studies show that expression of the TWEAK receptor, Fn14, increases in some of these same diseases. On the other hand, we monitored our TWEAK-Tg mice for almost 1 year and found no indications of uncontrolled cell growth or frank neoplasia. Rather, the oval cell population reaches a plateau in which hyperplasia appears to be balanced by apoptosis.
In summary, our study reveals a novel role for TWEAK: initiation of oval cell proliferation, most likely by directly signaling to these Fn14 receptor–bearing cells. Unlike other growth factors and cytokines that play a role in both oval cell and hepatocyte growth, TWEAK appears to have a selective effect on oval cells. The ability to block oval cell expansion in the DDC model by inhibiting the TWEAK pathway demonstrates that this inducible pathway is an important stimulator of hepatic stem cell responses during liver injury. Moreover, since TWEAK binds to many progenitor cells of the mesenchymal lineage, including human muscle satellite cells, preadipocytes, and chondrocytes (T. Zheng, unpublished observations), it is possible that TWEAK may be a regulator of organ progenitor cells in multiple organ systems. Its potential role in chronic human liver diseases in which progenitor cell activation is featured is suggested by the increased expression of Fn14 on the abundant duct-like structures present in alcoholic cirrhosis and viral hepatitis.