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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2010 Apr 19;107(18):8248–8253. doi: 10.1073/pnas.0912203107

The Hippo–Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis

Kwang-Pyo Lee a,1, Joo-Hyeon Lee a,1, Tae-Shin Kim a, Tack-Hoon Kim a, Hee-Dong Park a, Jin-Seok Byun b, Min-Chul Kim a, Won-Il Jeong b, Diego F Calvisi c, Jin-Man Kim d, Dae-Sik Lim a,2
PMCID: PMC2889558  PMID: 20404163

Abstract

Loss of Hippo signaling in Drosophila leads to tissue overgrowth as a result of increased cell proliferation and decreased cell death. YAP (a homolog of Drosophila Yorkie and target of the Hippo pathway) was recently implicated in control of organ size, epithelial tissue development, and tumorigenesis in mammals. However, the role of the mammalian Hippo pathway in such regulation has remained unclear. We now show that mice with liver-specific ablation of WW45 (a homolog of Drosophila Salvador and adaptor for the Hippo kinase) manifest increased liver size and expansion of hepatic progenitor cells (oval cells) and eventually develop hepatomas. Moreover, ablation of WW45 increased the abundance of YAP and induced its localization to the nucleus in oval cells, likely accounting for their increased proliferative capacity, but not in hepatocytes. Liver tumors that developed in mice heterozygous for WW45 deletion or with liver-specific WW45 ablation showed a mixed pathology combining characteristics of hepatocellular carcinoma and cholangiocarcinoma and seemed to originate from oval cells. Together, our results suggest that the mammalian Hippo–Salvador pathway restricts the proliferation of hepatic oval cells and thereby controls liver size and prevents the development of oval cell–derived tumors.

Keywords: conditional knockout mice, DDC diet, hepatoma, WW45, YAP

The mammalian Hippo signaling pathway has been implicated in regulation of contact inhibition, organ size, and tumorigenesis (1–4). Such regulation is thought to be mediated by control of the expression level or localization of YAP, a major target of the Hippo pathway. YAP is overexpressed in certain mammalian cancers, and YAP transgenic mice show increased liver size and intestinal dysplasia and eventually develop liver tumors. The role of YAP in control of organ size and tumorigenesis prompted us to examine whether upstream components of the Hippo pathway indeed function to regulate YAP in this context. However, embryonic mortality (WW45−/−, LATS2−/−, MST1−/−MST2−/−, or YAP−/−) or the absence of any overt enlargement of specific organs (LATS1−/−) in mice lacking such components has hampered this investigation (5–9). The generation of conditional knockout mice would thus seem to be warranted for investigation of the role of the mammalian Hippo pathway in the control of liver size and tumorigenesis.

Primary liver tumors have been categorized into two major types: hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), which originate from hepatocytes and cholangiocytes, respectively. However, some primary hepatomas exhibit an intermediate or combined (HCC/CC) phenotype and are thought to be derived from transformed progenitor (oval) cells or by dedifferentiation of mature cells (10–16). Oval cells are thought to be bipotential progenitor cells that can differentiate into either hepatocytes or ductal cholangiocytes but do so only if proliferation of hepatocytes is inhibited (17–19). However, the precise mechanism responsible for regulation of oval cell proliferation and how its deregulation contributes to tumor development remain poorly understood.

The mammalian Hippo pathway is thought to play an important role in regulation of various types of progenitor cells. Epithelial tissues of WW45 null embryos and the intestine of YAP transgenic mice manifest hyperplasia and dysplasia associated with expansion of progenitor cells (1, 5). We have now generated conditional knockout mice in which the gene for WW45, a homolog of Drosophila Salvador (Sav), is inactivated specifically in the liver. We characterized the role of the mammalian Hippo–Sav pathway in regulation of hepatic progenitor (oval) cell proliferation, liver size, and tumorigenesis with the use of these mice as well as of WW45+/− mice.

Results

Liver-Specific Ablation of WW45 Results in Liver Enlargement and Expansion of Hepatic Progenitor Cells.

To determine the role of the mammalian Hippo pathway, we have generated the liver-specific WW45 knockout mouse with the use of the Albumin-Cre mice (WW45flox/floxAlbumin-Cre mice, hereafter designated WW45 Liv-cKO) (Fig. S1). The liver of WW45 Liv-cKO mice was significantly larger than that of control animals (Fig. 1A). Although it exhibited regions of abnormal morphology with irregular and enlarged hepatocytes, the gross architecture of the liver of WW45 Liv-cKO mice was normal (Fig. S2A). In addition, we did not see any overt defects in the maturation or differentiation of hepatocytes (Fig. S2B) or any malfunction of the liver (Fig. S2C and Table S1) in WW45 Liv-cKO mice.

Fig. 1.

Fig. 1.

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Abnormal expansion of A6-positive oval cells in the liver of WW45 Liv-cKO mice. (A) Representative livers of 6-month-old WW45 Liv-cKO and control (Ctrl) mice (Upper). Liver weight as a percentage of body weight was also measured for mice (n ≥ 7) at the indicated ages (Lower). **P < 0.01; ***P < 0.001. (B) Liver sections from 6-month-old control and WW45 Liv-cKO mice were stained with H&E (Upper), revealing an increased number of immature progenitor cells (arrowheads) in the mutant mice. Such sections were also stained with antibodies to A6 (green) and to pan-CK (red), revealing colocalization of both antigens in oval cells. PT, portal tract; CV, central vein. (C) Evaluation of cell proliferation by immunofluorescence analysis with antibodies to PCNA (red) and to pan-CK (green) in liver sections of WW45 Liv-cKO and control mice at the indicated ages (Upper). White dotted lines, portal tract; yellow dotted lines, central vein. The percentages of pan-CK–negative parenchymal cells or pan-CK–positive oval cells that were positive for PCNA were determined (Lower). **P < 0.01; ***P < 0.001 (n = 4). (Scale bars, 100 μm in B; 200 μm in C.)

By 6 months of age, however, WW45 Liv-cKO mice manifested a marked increase in the number of immature progenitor cells, or oval-like cells, in the liver, compared with control animals. To confirm that these immature cells were true oval cells, we performed immunostaining analysis for marker proteins. The A6 antigen and various cytokeratins (CKs), such as CK8 and CK19, are specifically expressed in proliferating oval cells and normal biliary epithelial cells (20–23). The liver of WW45 Liv-cKO mice exhibited an increased number of cells positive for both A6 and CK expression around portal tracts (Fig. 1B). Staining for the hematopoietic marker CD34, which is expressed in proliferating oval cells (24), also confirmed oval cell expansion in WW45 Liv-cKO (Fig. S2D). We next analyzed the number of proliferating cell nuclear antigen (PCNA)-positive cells in mice to determine the proliferative potential of oval cells. The percentage of CK-positive cells that were also positive for PCNA was significantly greater for WW45 Liv-cKO mice at 3–12 months of age than for control mice, whereas the proliferative index of CK-negative parenchymal cells was similar for mutant and control animals (Fig. 1C). These results thus indicated that deletion of WW45 in the liver results in the specific proliferation and expansion of oval cells.

To determine whether oval cell expansion in the mutant mice was secondary to inherent liver damage, we examined the effect of partial hepatectomy. After partial hepatectomy, the healthy liver regenerates solely through hepatocyte proliferation (25). Only when hepatocytes are not able to restore the damaged parenchyma sufficiently does the liver depend on oval cells for regeneration (18, 26). The regeneration capacity of the liver of WW45 Liv-cKO mice seemed normal through completion of liver recovery (Fig. S3). Moreover, we did not detect any further increase in the number of A6-positive oval cells in the liver of the mutant mice during liver regeneration. Thus, A6-positive oval cell expansion in the mutant mice results from an intrinsic genetic defect rather than from hepatocyte damage or impaired hepatic regeneration.

DDC Treatment Increases Oval Cell Number and Liver Size in WW45 Liv-cKO Mice.

We next investigated whether there might be a direct link between oval cell proliferation and liver size with the use of a model of liver injury. A diet supplemented with 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), a porphyrinogenic hepatotoxin, induces the proliferation of oval cells in the region of portal tracts (19, 27, 28). We first examined liver size in mice fed a diet containing 0.1% DDC. Liver weight as a percentage of body weight increased to a greater extent in WW45 Liv-cKO mice than in control mice (Fig. 2A). The absolute size of the liver of the mutant mice was also markedly greater than that of control mice at 6 weeks after initiation of DDC treatment (Fig. 2B). By 7 days after the onset of the DDC diet, the liver began to exhibit structural changes associated with oval cell proliferation and atypical ductal proliferation formation in the vicinity of portal tracts in both control and mutant mice (Fig. S4A). BrdU labeling, however, revealed a marked increase in the number of proliferating A6-positive cells in the mutant mice (Fig. S4B). By 2–4 weeks, the structural changes in the portal tract regions were more pronounced in the mutant mice. The A6-positive area of the mutant mice was thus substantially larger than that in control mice (Fig. 2C and D and Fig. S4C). However, we did not see any difference in the number of apoptotic cells in parenchymal regions in serial sections of the liver between WW45 Liv-cKO and control mice (Fig. S4D), indicating that the greater expansion of A6-positive oval cells in the mutant mice did not result from an increased hepatocyte sensitivity to DDC. These results thus suggested that DDC induced an earlier and more efficient expansion of oval cells, resulting in rapid and pronounced enlargement of the liver, in WW45 Liv-cKO mice.

Fig. 2.

Fig. 2.

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Increased proliferative response of A6-positive oval cells to DDC treatment in WW45 Liv-cKO mice. (A) Five-week-old WW45 Liv-cKO or control mice (n = 5) were fed a diet containing 0.1% DDC for the indicated times, after which liver weight as a percentage of body weight was measured. *P < 0.05. (B) Livers from control and WW45 Liv-cKO mice fed a 0.1% DDC diet for 6 weeks. (C) Liver sections from control or WW45 Liv-cKO mice fed a 0.1% DDC diet for 4 weeks were subjected to H&E staining or to immunohistochemical staining for A6, as indicated. Asterisks demarcate porphyrin accumulation. (D) The area of A6 staining in liver sections was quantitated with the use of ImageJ software for mutant and control mice fed a 0.1% DDC diet for the indicated times. Data are expressed as a percentage of thresholded areas. *P < 0.05 (n ≥ 3). (E) Oval cells isolated from DDC-treated control or WW45 Liv-cKO mice were incubated in growth medium containing 10 μM BrdU for 5 h. The cells were then subjected to immunofluorescence analysis (Upper) with antibodies to A6 (green) and to BrdU (red), and nuclei were stained with DAPI (blue). The percentage of A6-positive cells that were also positive for BrdU was determined for individual colonies (Lower). *P < 0.05 (n = 26 colonies, control; n = 22 colonies, mutant). (F) Oval cells isolated from DDC-treated control or WW45 Liv-cKO mice were cultured in growth medium for 1 week and then subjected to immunofluorescence analysis (Upper) with antibodies to A6 (green) and to Ki67 (red); nuclei were stained with DAPI (blue). DIC images are shown in the top four panels. The number of DAPI-stained nuclei per colony was also determined (Lower). Data are presented as a box-and-whisker plot for 33 (control) or 28 (mutant) colonies. ***P < 0.001. (Scale bars, 200 μm in C; 50 μm in E and F.)

Increased Proliferative Capacity of Oval Cells Isolated from WW45 Liv-cKO Mice.

We next examined the proliferative capacity of oval cells isolated from mice fed a diet containing 0.1% DDC for 3 weeks. The cells proliferated and formed colonies within 1 week in vitro. Consistent with the in vivo data, both BrdU labeling and Ki67 staining revealed that the proliferative capacity of the mutant oval cells was significantly greater than that of the control cells (Fig. 2 E and F). Furthermore, the number of cells per colony and colony size were markedly greater for the mutant oval cells than for the control cells. These data thus suggested that WW45-deficient oval cells have an intrinsically increased proliferative capacity independent of environmental factors.

WW45 Ablation in the Liver Results in Deregulation of YAP.

The phenotype of WW45 Liv-cKO mice was markedly similar to that of mice specifically overexpressing YAP in the liver (1, 2). We therefore examined whether YAP might be up-regulated in WW45 Liv-cKO mice. YAP began to accumulate in the liver of WW45 Liv-cKO mice by 3 months of age, a time at which oval cells begin to expand spontaneously. DDC-induced oval cell expansion was also associated with a marked increase in YAP expression in the liver of WW45 Liv-cKO mice but not in that of control mice (Fig. 3A). These results thus suggested that the Hippo pathway limits hyperexpansion of the oval cell population, possibly through regulation of YAP abundance.

Fig. 3.

Fig. 3.

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Deregulation of YAP as a result of liver-specific ablation of WW45. (A) Liver lysates prepared from control (C) and WW45 Liv-cKO (cKO) mice either at the indicated ages (Left) or after maintenance on a 0.1% DDC diet for the indicated times (Right) were subjected to immunoblot analysis with antibodies to the indicated proteins, with GAPDH examined as a loading control. FL and NT, full-length and NH2-terminal fragment of MST1, respectively. (B) Nuclear (N) and cytosolic (C) fractions of the liver of 6-month-old (Upper) or DDC-treated (Lower) control and WW45 Liv-cKO mice were subjected to immunoblot analysis. Lamin B and α-tubulin were examined as nuclear and cytosolic marker proteins, respectively. (C) Liver sections of control and WW45 Liv-cKO mice either at 6 months of age or maintained on a 0.1% DDC diet for 4 weeks were stained with antibodies to YAP and to A6. Arrowheads, periductal oval cells; asterisks demarcate porphyrin accumulation. (D) Excision PCR analysis of hepatocytes and an oval cell–enriched fraction (OEF) that were derived from WT or WW45flox/flox (f/f) mice and infected with the Adeno-Cre virus (Upper). Arrow indicates the position of products (165 bp) derived from the excised WW45 allele. Lysates of such infected cells were also subjected to immunoblot analysis (Lower). Asterisks indicate nonspecific bands. (E) Oval cells isolated from the liver of DDC-treated control or WW45 Liv-cKO mice were subjected to immunofluorescence staining for A6 (green) and YAP (red) as well as to staining with DAPI (blue). (F) Oval cells isolated from the liver of DDC-treated WT or WW45flox/flox mice were infected with Adeno-Cre and then subjected to immunofluorescence staining for A6 (green) and YAP (red) as well as to staining with DAPI (blue). (G) Oval cells isolated and infected as in F were incubated in growth medium containing 10 μM BrdU for 5 h. The cells were then stained with antibodies to A6 and to BrdU, and nuclei were stained with DAPI. The percentage of A6-positive cells that were also positive for BrdU was determined for individual colonies (WT, n = 21; WW45 flox/flox, n = 19). *P < 0.05. (Scale bars, 100 μm in C; 50 μm in E; 20 μm in F.)

The Hippo pathway inhibits YAP activity by mediating YAP phosphorylation and its consequent retention in the cytoplasm (4, 5, 29–33). We therefore analyzed the phosphorylation status of YAP and other Hippo pathway components. Unexpectedly, phosphorylation of YAP was increased in the liver of aged or DDC-treated WW45 Liv-cKO mice relative to that in the liver of corresponding control animals. This increased phosphorylation of YAP was accompanied by up-regulation of the expression of other Hippo pathway components, including MST1, LATS1, and LATS2 (Fig. 3A). To provide insight into these paradoxical results, we evaluated the activity of YAP by examining its subcellular localization. Subcellular fractionation showed that the amount of YAP in the nuclear fraction was increased in the liver of aged or DDC-treated WW45 Liv-cKO mice (Fig. 3B). Increased nuclear localization of YAP was also apparent in periportal oval cells of such WW45 Liv-cKO mice compared with control animals (Fig. 3C). The abundance of cyclin D1, which is encoded by a YAP target gene, was also increased in the liver of aged mutant mice (Fig. 3A). Thus, despite the observation that the level of phosphorylated YAP and of other Hippo pathway components was increased, these results indicate that YAP is hyperactivated in the liver of WW45 Liv-cKO mice.

Given that we observed an oval cell–specific increase in proliferative index in WW45 Liv-cKO mice (Fig. 1C), we examined whether these alterations in the Hippo pathway and the resulting hyperactivation of YAP are also specific to oval cells. Consistent with this notion, we detected accumulation of Hippo pathway components including YAP in an oval cell–enriched fraction of WW45 Liv-cKO mice. In contrast, such accumulation was minimal in a hepatocyte fraction of WW45 Liv-cKO mice (Fig. S5A). To determine more accurately the early response to WW45 ablation in the liver and exclude confounding factors associated with development, we inactivated WW45 with the use of the Adeno-Cre system in isolated hepatocytes and oval cells. Deletion of WW45 and the absence of WW45 protein in such cells were confirmed by PCR and immunoblot analyses, respectively (Fig. 3D). The abundance of YAP and other Hippo pathway components was increased in these WW45-deficient oval cells but not in the mutant hepatocytes. Isolated oval cells from the mutant mice showed intense nuclear staining for YAP, whereas those isolated from control mice showed a diffuse pattern of YAP immunoreactivity in the cytoplasm (Fig. 3E and Fig. S5B). In addition, isolated oval cells subjected to ablation of WW45 with the Adeno-Cre system also showed increased nuclear staining for YAP (Fig. 3F) and an increased proliferative capacity (Fig. 3G) compared with control cells. Consistent with oval cell–specific accumulation of YAP, we also found that connective tissue growth factor (ctgf) and birc5 (survivin), the known YAP target genes (2, 34), were significantly up-regulated in oval cell–enriched fraction but not in isolated hepatocytes (Fig. S5C). These results supported the notion that nuclear localization and hyperactivation of YAP in oval cells underlie the expansion of these cells in WW45 Liv-cKO mice.

Development of Mixed-Type Liver Tumors in WW45+/− and WW45 Liv-cKO Mice.

Because WW45 heterozygous (WW45+/−) mice developed normally, we next evaluated possible effect of haploinsufficiency of WW45 on tumorigenesis. Seventy-four percent of WW45+/− mice developed liver tumors, and all three surviving WW45 knockout mice examined also developed liver tumors and died earlier than did heterozygotes (Fig. S6A). Tumor nodules of hepatomas were detected in both WW45+/− and WW45 Liv-cKO mice by 12 months of age (Fig. S6B). Consistent with the WW45 Liv-cKO mice, we observed up-regulation of A6 expression both in the periportal regions of the liver of WW45+/− mice as early as at 8 months of age as well as in hepatomas of such animals at 12 or 18 months of age (Fig. 4A). Furthermore, almost all hepatomas that developed in WW45+/− or WW45 Liv-cKO mice had an intermediate phenotype (Fig. 4B), suggesting that liver tumorigenesis can be initiated by hepatic progenitor (oval) cells (35–37).

Fig. 4.

Fig. 4.

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Mixed-type liver tumor development in WW45 mutant mice. (A) Liver or hepatoma sections of WW45+/− mice at 8 or at 12 or 18 months of age, respectively, were subjected to H&E staining and to immunohistochemical staining for A6. (Insets) Small A6-positive oval cells in hepatic cords at a magnification twice that for the main panels. (B) Hepatoma sections from WW45+/− or WW45 Liv-cKO mice at 14 months of age were subjected to H&E staining and to immunohistochemical staining for A6. Strands or trabeculae of small A6-positive oval cells were distributed within the hepatomas (a, a′, c, and c′). A6-positive oval cells were also organized in glandlike structures within the tumors (b, b′, d, and d′). (C) Sections of normal liver or hepatoma tissue from WW45+/+ or WW45+/− mice, respectively, were stained with antibodies to YAP. (D) Immunoblot analysis of tumors (T) at initiation, early, or advanced (Adv) stages and of corresponding nontumor (N) regions in the liver of WW45+/− mice as well as of tumor regions (T1, T2) and a nontumorous region (N) from the same liver of a WW45 Liv-cKO mouse. (Scale bars, 200 μm in A; 100 μm in B; 50 μm in C.)

Finally, we examined possible changes in YAP localization and abundance in association with liver tumorigenesis. Hepatoma tissue from WW45+/− mice showed nuclear localization of YAP (Fig. 4C), and liver tumors from WW45+/− or WW45 Liv-cKO mice also contained increased amounts of YAP that seemed to correlate with tumor progression (Fig. 4D), highlighting the importance of YAP abundance in liver tumorigenesis induced by ablation of WW45. We also observed accumulation of Hippo pathway components including phosphorylated YAP in hepatomas of the mutant mice. However, this up-regulation seemed insufficient to suppress oncogenic activity of YAP, as revealed by YAP localization and eventual tumorigenesis. These results thus provide evidence that WW45 acts to suppress oval cell expansion and liver tumorigenesis by inhibiting the accumulation and activation of YAP.

Discussion

We previously showed that WW45 suppresses expansion of undifferentiated epithelial cells, including keratinocytes (5). However, the relevance of WW45 in progenitor cells of adults and in tumorigenesis remained unexamined. We have now generated mice with liver-specific ablation of WW45 to address this issue. A recent study has underscored the importance of the Hippo pathway in control of liver size and tumorigenesis via regulation of the proliferation of differentiated hepatocytes (38). However, given that YAP, a downstream effector of the Hippo pathway, contributes to expansion of stem or progenitor cells (1), it remained possible that dysregulation of the Hippo pathway in the liver might result in inappropriate expansion of liver progenitor cells. We have now found that the mammalian Hippo–Sav pathway regulates homeostasis of progenitor (oval) cells in the adult liver. WW45 Liv-cKO mice exhibited pronounced overgrowth of the liver. Although this characteristic is similar to that of mice with liver-specific knockout of MST1 and MST2, mammalian homologs of Drosophila Hippo, the overproliferation of oval cells, not that of hepatocytes, contributes to such overgrowth in WW45 Liv-cKO mice. We thus found that the increase in liver size induced by exposure to DDC was also enhanced in these animals compared with that in control mice. Finally, WW45 Liv-cKO mice developed tumors with a mixed (HCC/CC) phenotype, with such tumors being thought to originate from transformed oval cells (13, 14, 16). These tumors are thus distinct from the HCC, originating from aberrant proliferation of hepatocytes, that was observed in the MST1/2 conditional knockout mice (38). Our present findings therefore provide evidence that the mammalian Hippo–Sav pathway restricts the proliferation not only of epithelial progenitor cells during embryonic development but also of oval cells in the adult liver.

YAP is frequently overexpressed in HCC (39), and liver-specific overexpression of YAP results in enlargement of the liver and the development of liver tumors (2). YAP overexpression acting in cooperation with cIAP1 and c-Myc in hepatic progenitor cells also leads to tumorigenesis (40). Consistent with the notion that the Hippo pathway is the major suppressive regulator of YAP, ablation of WW45 in the liver also induces the formation of liver tumors. We found that even liver tumors isolated from WW45+/− mice, which retained the wild-type WW45 allele, manifested substantial down-regulation of WW45 protein (Fig. S6C), suggesting that the remaining wild-type WW45 allele may be silenced, possibly as a result of promoter methylation. WW45 Liv-cKO mice also exhibited pronounced accumulation of YAP in the liver. However, this accumulation of YAP was accompanied by that of other Hippo pathway components and phosphorylated YAP. Nevertheless, a substantial amount of YAP was localized to nuclei in the mutant liver. Such localization was most evident in mutant oval cells, and these cells accordingly showed an increased proliferation index. Furthermore, liver tumors in the mutant mice were enriched in transformed oval cells. We therefore propose that the ablation of WW45 results in hyperactivation of YAP, preferentially in oval cells, and thereby promotes liver tumorigenesis.

The cleaved forms of MST1 and MST2 were recently shown to be required for maintenance of hepatocyte quiescence in the adult liver (38). Cleaved MST1/2, in contrast to the full-length proteins, cannot interact with WW45 as a result of loss of the SARAH domain. It was therefore suggested that, whereas cleaved MST1/2 may be a key regulator of YAP in differentiated hepatocytes, WW45 may play a minimal role. Although our data suggest that the cleaved form of MST1 may not be the major form of MST1 in hepatocytes (Fig. 3A), they nevertheless indicate that WW45 indeed plays a minor role in differentiated hepatocytes. Hepatocyte fractions lacking WW45 thus showed only marginal, if any, up-regulation of YAP. WW45 Liv-cKO mouse hepatocytes also showed a proliferative index similar to that of control hepatocytes. An acute increase in liver size and tumorigenesis due to proliferation of hepatocytes, similar to those apparent in both YAP transgenic mice (1, 2) and MST1/2 conditional knockout mice (38), were not observed in the WW45 mutant liver. These results suggest that WW45 plays a minor role in maintenance of hepatocyte quiescence.

In contrast, we observed accumulation of YAP and other Hippo pathway components in the oval cell fraction of the WW45-deficient liver. Accordingly, we found that ctgf and birc5, which were previously shown to be up-regulated in the liver of YAP transgenic mice (2) and MCF10A YAP-stable cell line (34), were specifically induced in oval cell–enriched fraction but not in isolated hepatocytes. These findings suggest that WW45 in oval cells, unlike that in hepatocytes, plays a key role in the regulation of YAP. Consistently, we found that the proliferation index of WW45-deficient oval cells was increased both in vivo and in vitro. WW45 deficiency resulted in the late development of mixed-type liver tumors indicative of oval cell expansion. We therefore suggest that two distinct upstream regulators of YAP operate in differentiated hepatocytes and in oval cells. In hepatocytes, MST1/2 (Hippo) regulate YAP in a WW45 (Sav)–independent manner, whereas in oval cells, WW45 (Sav) is an essential regulator of YAP. The Hippo–Sav pathway is thus required to restrict the proliferation of adult liver stem or progenitor cells. On the basis of our present data and our previous results with keratinocytes (5), it is possible that WW45 functions as the major suppressor of YAP in stem or progenitor cells in general and thereby inhibits the inappropriate expansion of undifferentiated cells. In fact, recent reports suggested that the mammalian hippo pathway might be required to repress the oval cell activation (41, 42), supporting the hypothesis that Hippo signaling directly suppresses the proliferation of liver progenitor cells (oval cells).

Although all Hippo pathway components were found to be up-regulated in the WW45 Liv-cKO liver, we did not detect an increase in the abundance of the corresponding mRNAs. Such an increase in mRNA levels was apparent only after tumor development in WW45+/− mice, although the increase was not as pronounced as that in protein levels (Fig. S6D). Regulation by stabilization of Hippo pathway components therefore seems to be operative in normal liver tissue, with feedback regulation of such components at the transcriptional level possibly operating only in the tumor environment. Further investigation of such regulatory mechanisms may provide important insight into homeostasis of Hippo signaling output.

Materials and Methods

Generation of WW45 Liv-cKO Mice.

Methods for generation of WW45 Liv-cKO mice are described in SI Materials and Methods.

DDC Feeding Protocol.

For induction of oval cell proliferation, mice at ≈5 weeks of age were fed a diet comprising standard chow supplemented with 0.1% DDC (Sigma-Aldrich) (28).

Histologic Analysis.

Liver tissue was fixed in 4% paraformaldehyde for preparation of frozen sections and in 10% formalin for paraffin sections. Detailed protocols and all antibodies for staining are described in SI Materials and Methods.

Isolation and Analysis of Hepatocyte and Oval Cell–Enriched Fractions.

Hepatocytes were prepared by a standard two-step perfusion protocol (43). For isolation of oval cells, mice at 6–8 weeks of age were first fed a diet containing 0.1% DDC for 3 weeks, after which the liver was perfused in situ via the hepatic portal vein as previously described (44). Methods for isolation and analysis of these cells are described in SI Materials and Methods.

Adeno-Cre Infection.

Isolated primary hepatocytes and oval cells were incubated for 48 h with Adeno-Cre (kindly provided E.-J. Lee) at a multiplicity of infection of 100 pfu per cell (45). The cells were then exposed to fresh growth medium and subjected to immunoblot or immunofluoresence analysis or to assay of BrdU incorporation.

Immunoblot Analysis and Subcellular Fractionation.

Liver tissue as well as isolated hepatocyte or oval cell–enriched fractions were homogenized in Proprep lysis buffer (iNtRON Biotechnology) containing protease and phophatase inhibitors. The lysates were centrifuged at 15,000 × g for 20 min at 4 °C, and the resulting supernatants were subjected to immunoblot analysis. The antibodies used for detection are described in SI Materials and Methods. Nuclear and cytosolic extracts were prepared with the use of NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology).

Statistical Analysis.

Quantitative data are presented as means ± SD unless indicated otherwise. Differences between means were evaluated by Student's unpaired t test. P < 0.05 was considered statistically significant.

Supplementary Material

Supporting Information
supp_107_18_8248__index.html (947B, html)

Acknowledgments

We thank V. Factor (National Cancer Institute, Bethesda, MD) for kindly providing antibodies to A6; E.-J. Lee (Yonsei University, College of Medicine, Seoul, Korea) for providing Adeno-Cre; and Randy Johnson and Yingzi Yang for sharing results before publication. This study was supported by grants from the National Research Laboratory Program and the Korea National Cancer Center Program.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0912203107/-/DCSupplemental.

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