The Hippo Salvador pathway restrains hepatic oval cell ... · The Hippo–Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis Kwang-Pyo

The Hippo–Salvador pathway restrains hepatic ovalcell proliferation, liver size, and liver tumorigenesisKwang-Pyo Leea,1, Joo-Hyeon Leea,1, Tae-Shin Kima, Tack-Hoon Kima, Hee-Dong Parka, Jin-Seok Byunb, Min-Chul Kima,Won-Il Jeongb, Diego F. Calvisic, Jin-Man Kimd, and Dae-Sik Lima,2

aNational Creative Research Initiatives Center, Department of Biological Sciences and bGraduate School of Medical Science and Engineering, BiomedicalResearch Center, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea; cInstitut für Pathologie, Ernst-Moritz-Arndt-Universität,Greifswald 17487, Germany; and dDepartment of Pathology, College of Medicine, Chungnam National University, Daejeon 301-721, Korea

Edited by Tyler Jacks, Massachusetts Institute of Technology, Cambridge, MA, and approved March 31, 2010 (received for review October 23, 2009)

Loss of Hippo signaling in Drosophila leads to tissue overgrowth asa 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 tissuedevelopment, and tumorigenesis in mammals. However, the roleof the mammalian Hippo pathway in such regulation has remainedunclear. We now show that mice with liver-specific ablation ofWW45 (a homolog of Drosophila Salvador and adaptor for theHippokinase)manifest increased liver size andexpansionof hepaticprogenitor cells (oval cells) and eventually develop hepatomas.Moreover, ablation of WW45 increased the abundance of YAP andinduced its localization to the nucleus in oval cells, likely accountingfor their increased proliferative capacity, but not in hepatocytes.Liver tumors that developed in mice heterozygous for WW45 de-letion or with liver-specificWW45 ablation showed amixed pathol-ogy combining characteristics of hepatocellular carcinoma andcholangiocarcinoma and seemed to originate from oval cells. To-gether, our results suggest that the mammalian Hippo–Salvadorpathway restricts the proliferation of hepatic oval cells and therebycontrols liver size and prevents the development of oval cell–derived tumors.

conditional knockout mice | DDC diet | hepatoma | WW45 | YAP

The mammalianHippo signaling pathway has been implicated inregulation of contact inhibition, organ size, and tumorigenesis

(1–4). Such regulation is thought to be mediated by control of theexpression level or localization ofYAP, amajor target of theHippopathway. YAP is overexpressed in certain mammalian cancers, andYAP transgenic mice show increased liver size and intestinal dys-plasia and eventually develop liver tumors. The role of YAP incontrol of organ size and tumorigenesis prompted us to examinewhether upstream components of the Hippo pathway indeedfunction to regulate YAP in this context. However, embryonicmortality (WW45−/−, LATS2−/−, MST1−/−MST2−/−, or YAP−/−) orthe 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 thusseem to bewarranted for investigation of the role of themammalianHippo pathway in the control of liver size and tumorigenesis.Primary liver tumors have been categorized into twomajor types:

hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC),which originate from hepatocytes and cholangiocytes, respectively.However, some primary hepatomas exhibit an intermediate orcombined (HCC/CC)phenotypeandare thought to bederived fromtransformed progenitor (oval) cells or by dedifferentiation of ma-ture cells (10–16). Oval cells are thought to be bipotential pro-genitor cells that can differentiate into either hepatocytes or ductalcholangiocytes but do so only if proliferation of hepatocytes isinhibited (17–19). However, the precise mechanism responsible forregulation of oval cell proliferation and how its deregulation con-tributes 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 tis-

suesofWW45null embryos and the intestineofYAPtransgenicmicemanifest hyperplasia and dysplasia associated with expansion ofprogenitor cells (1, 5).Wehavenowgenerated conditional knockoutmice inwhich the gene forWW45, ahomologofDrosophilaSalvador(Sav), is inactivated specifically in the liver.Wecharacterized the roleof the mammalian Hippo–Sav pathway in regulation of hepaticprogenitor (oval) cell proliferation, liver size, and tumorigenesiswiththe use of these mice as well as ofWW45+/− mice.

ResultsLiver-Specific Ablation of WW45 Results in Liver Enlargement andExpansion of Hepatic Progenitor Cells. To determine the role of themammalian Hippo pathway, we have generated the liver-specificWW45 knockout mouse with the use of the Albumin-Cre mice(WW45flox/floxAlbumin-Cremice, hereafter designated WW45 Liv-cKO) (Fig. S1). The liver ofWW45 Liv-cKOmice was significantlylarger than that of control animals (Fig. 1A). Although it exhibitedregions of abnormal morphology with irregular and enlargedhepatocytes, the gross architecture of the liver ofWW45 Liv-cKOmice was normal (Fig. S2A). In addition, we did not see any overtdefects in the maturation or differentiation of hepatocytes (Fig.S2B) or any malfunction of the liver (Fig. S2C and Table S1) inWW45 Liv-cKO mice.By 6 months of age, however,WW45 Liv-cKO mice manifested

a marked increase in the number of immature progenitor cells, oroval-like cells, in the liver, compared with control animals. Toconfirm that these immature cells were true oval cells, we per-formed immunostaining analysis for marker proteins. The A6antigen and various cytokeratins (CKs), such as CK8 and CK19,are specifically expressed in proliferating oval cells and normalbiliary epithelial cells (20–23). The liver of WW45 Liv-cKO miceexhibited an increased number of cells positive for bothA6andCKexpression around portal tracts (Fig. 1B). Staining for the hema-topoietic marker CD34, which is expressed in proliferating ovalcells (24), also confirmed oval cell expansion in WW45 Liv-cKO(Fig. S2D). We next analyzed the number of proliferating cellnuclear antigen (PCNA)-positive cells in mice to determine theproliferative potential of oval cells. The percentage of CK-positivecells that were also positive for PCNAwas significantly greater forWW45 Liv-cKOmice at 3–12 months of age than for control mice,whereas the proliferative index of CK-negative parenchymal cellswas similar formutant and control animals (Fig. 1C). These results

Author contributions: K.-P.L., J.-H.L., and D.-S.L. designed research; K.-P.L., J.-H.L., T.-S.K.,T.-H.K., H.-D.P., J.-S.B., M.-C.K., W.-I.J., D.F.C., J.-M.K., and D.-S.L. performed research;K.-P.L., J.-H.L., T.-S.K., T.-H.K., H.-D.P., J.-S.B., M.-C.K., W.-I.J., D.F.C., J.-M.K., and D.-S.L.analyzed data; and K.-P.L., J.-H.L., and D.-S.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1K.-P.L. and J.-H.L. contributed equally to this work.2To whom correspondence should be addressed. E-mail: [email protected].

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

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thus indicated that deletion of WW45 in the liver results in thespecific 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 ofpartial hepatectomy. After partial hepatectomy, the healthy liverregenerates solely through hepatocyte proliferation (25). Onlywhenhepatocytes are not able to restore the damagedparenchymasufficiently does the liver dependonoval cells for regeneration (18,26). The regeneration capacity of the liver ofWW45Liv-cKOmiceseemed normal through completion of liver recovery (Fig. S3).Moreover, we did not detect any further increase in the number ofA6-positive oval cells in the liver of the mutant mice during liverregeneration. Thus, A6-positive oval cell expansion in the mutantmice results from an intrinsic genetic defect rather than from he-patocyte damage or impaired hepatic regeneration.

DDC Treatment Increases Oval Cell Number and Liver Size in WW45Liv-cKO Mice. We next investigated whether there might be a directlink between oval cell proliferation and liver size with the use ofa model of liver injury. A diet supplemented with 0.1% 3,5-dieth-oxycarbonyl-1,4-dihydrocollidine (DDC), a porphyrinogenic hep-atotoxin, induces theproliferationof oval cells in the regionof portaltracts (19, 27, 28). We first examined liver size in mice fed a dietcontaining 0.1%DDC. Liver weight as a percentage of body weightincreased to a greater extent inWW45Liv-cKOmice than in controlmice (Fig. 2A). The absolute size of the liver of themutantmice wasalso markedly greater than that of control mice at 6 weeks afterinitiation of DDC treatment (Fig. 2B). By 7 days after the onset ofthe DDC diet, the liver began to exhibit structural changes associ-ated with oval cell proliferation and atypical ductal proliferationformation in the vicinity of portal tracts in both control and mutantmice (Fig. S4A). BrdU labeling, however, revealed a marked in-crease in the number of proliferatingA6-positive cells in themutantmice (Fig. S4B). By 2–4 weeks, the structural changes in the portaltract regions were more pronounced in the mutant mice. The A6-positive area of the mutant mice was thus substantially larger thanthat in control mice (Fig. 2C andD and Fig. S4C). However, we didnot see any difference in the number of apoptotic cells in paren-chymal regions in serial sections of the liver between WW45 Liv-cKO and control mice (Fig. S4D), indicating that the greater ex-pansion of A6-positive oval cells in the mutant mice did not resultfrom an increased hepatocyte sensitivity toDDC.These results thussuggested that DDC induced an earlier and more efficient expan-sion of oval cells, resulting in rapid and pronounced enlargement ofthe liver, inWW45 Liv-cKO mice.

Increased Proliferative Capacity of Oval Cells Isolated fromWW45 Liv-cKO Mice.Wenext examined the proliferative capacity of oval cellsisolated 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 Ki67staining revealed that the proliferative capacity of the mutant ovalcells was significantly greater than that of the control cells (Fig. 2Eand F). Furthermore, the number of cells per colony and colonysize were markedly greater for the mutant oval cells than for thecontrol cells. These data thus suggested thatWW45-deficient ovalcells have an intrinsically increased proliferative capacity in-dependent of environmental factors.

WW45 Ablation in the Liver Results in Deregulation of YAP. Thephenotype ofWW45Liv-cKOmice was markedly similar to that ofmice specifically overexpressing YAP in the liver (1, 2). Wetherefore examinedwhetherYAPmight be up-regulated inWW45Liv-cKOmice.YAPbegan to accumulate in the liver ofWW45Liv-cKO mice by 3 months of age, a time at which oval cells begin toexpand spontaneously. DDC-induced oval cell expansion was alsoassociatedwith amarked increase inYAPexpression in the liver ofWW45 Liv-cKO mice but not in that of control mice (Fig. 3A).These results thus suggested that the Hippo pathway limitshyperexpansion of the oval cell population, possibly through reg-ulation of YAP abundance.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 ofYAP and other Hippo pathway components. Unexpectedly,phosphorylation of YAP was increased in the liver of aged orDDC-treated WW45 Liv-cKO mice relative to that in the liver ofcorresponding control animals. This increased phosphorylation ofYAPwas accompanied by up-regulation of the expression of otherHippo pathway components, including MST1, LATS1, andLATS2 (Fig. 3A). To provide insight into these paradoxical results,we evaluated the activity of YAP by examining its subcellular lo-calization. Subcellular fractionation showed that the amount ofYAP in the nuclear fraction was increased in the liver of aged or

Fig. 1. Abnormal expansion of A6-positive oval cells in the liver ofWW45 Liv-cKO mice. (A) Representative livers of 6-month-oldWW45 Liv-cKO and control(Ctrl) mice (Upper). Liver weight as a percentage of body weight was alsomeasuredformice (n≥7)at the indicatedages (Lower). **P<0.01; ***P<0.001.(B) Liver sections from 6-month-old control and WW45 Liv-cKO mice werestained with H&E (Upper), revealing an increased number of immature pro-genitor cells (arrowheads) in the mutant mice. Such sections were also stainedwith antibodies to A6 (green) and to pan-CK (red), revealing colocalization ofboth antigens in oval cells. PT, portal tract; CV, central vein. (C) Evaluation of cellproliferationby immunofluorescenceanalysiswithantibodies toPCNA(red) andto pan-CK (green) in liver sections of WW45 Liv-cKO and control mice at theindicated 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 PCNAwere determined (Lower). **P <0.01; ***P < 0.001 (n = 4). (Scale bars, 100 μm in B; 200 μm in C.)

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DDC-treated WW45 Liv-cKO mice (Fig. 3B). Increased nuclearlocalization of YAP was also apparent in periportal oval cells ofsuch WW45 Liv-cKO mice compared with control animals (Fig.3C). The abundance of cyclin D1, which is encoded by a YAPtarget gene, was also increased in the liver of aged mutant mice

(Fig. 3A). Thus, despite the observation that the level of phos-phorylated YAP and of other Hippo pathway components wasincreased, these results indicate that YAP is hyperactivated in theliver ofWW45 Liv-cKO mice.Given that we observed an oval cell–specific increase in pro-

liferative index in WW45 Liv-cKO mice (Fig. 1C), we examinedwhether these alterations in the Hippo pathway and the resultinghyperactivation of YAP are also specific to oval cells. Consistentwith this notion, we detected accumulation of Hippo pathwaycomponents including YAP in an oval cell–enriched fraction ofWW45Liv-cKOmice. In contrast, such accumulation wasminimalin a hepatocyte fraction of WW45 Liv-cKO mice (Fig. S5A). Todeterminemore accurately the early response toWW45 ablation inthe liver and exclude confounding factors associated with de-velopment, we inactivated WW45 with the use of the Adeno-Cresystem in isolated hepatocytes and oval cells. Deletion of WW45and the absence of WW45 protein in such cells were confirmed byPCR and immunoblot analyses, respectively (Fig. 3D). Theabundance of YAP and other Hippo pathway components wasincreased in theseWW45-deficient oval cells but not in themutanthepatocytes. Isolated oval cells from the mutant mice showed in-tense nuclear staining for YAP, whereas those isolated fromcontrolmice showed a diffuse pattern ofYAP immunoreactivity inthe cytoplasm (Fig. 3E and Fig. S5B). In addition, isolated ovalcells subjected to ablation of WW45 with the Adeno-Cre systemalso showed increased nuclear staining for YAP (Fig. 3F) and anincreased proliferative capacity (Fig. 3G) compared with controlcells. Consistent with oval cell–specific accumulation of YAP, wealso found that connective tissue growth factor (ctgf) and birc5(survivin), the known YAP target genes (2, 34), were significantlyup-regulated in oval cell–enriched fraction but not in isolatedhepatocytes (Fig. S5C). These results supported the notion thatnuclear localization and hyperactivation of YAP in oval cells un-derlie 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 de-veloped normally, we next evaluated possible effect of hap-loinsufficiency of WW45 on tumorigenesis. Seventy-four percentof WW45+/− mice developed liver tumors, and all three survivingWW45 knockout mice examined also developed liver tumors anddied earlier than did heterozygotes (Fig. S6A). Tumor nodules ofhepatomas were detected in both WW45+/− and WW45 Liv-cKOmice by 12 months of age (Fig. S6B). Consistent with the WW45Liv-cKOmice, we observed up-regulation ofA6 expression both inthe periportal regions of the liver ofWW45+/−mice as early as at 8months of age as well as in hepatomas of such animals at 12 or 18months of age (Fig. 4A). Furthermore, almost all hepatomas thatdeveloped in WW45+/− or WW45 Liv-cKO mice had an in-termediate phenotype (Fig. 4B), suggesting that liver tumorigen-esis can be initiated by hepatic progenitor (oval) cells (35–37).Finally, we examined possible changes in YAP localization and

abundance in associationwith liver tumorigenesis.Hepatoma tissuefromWW45+/−mice showed nuclear localization of YAP (Fig. 4C),and liver tumors fromWW45+/− orWW45 Liv-cKO mice also con-tained increased amounts of YAP that seemed to correlate withtumor progression (Fig. 4D), highlighting the importance of YAPabundance in liver tumorigenesis induced by ablation ofWW45.Wealso observed accumulation of Hippo pathway components in-cluding phosphorylated YAP in hepatomas of the mutant mice.However, this up-regulation seemed insufficient to suppress onco-genic activity of YAP, as revealed byYAP localization and eventualtumorigenesis. These results thus provide evidence thatWW45 actsto suppress oval cell expansion and liver tumorigenesis by inhibitingthe accumulation and activation of YAP.

Fig. 2. Increased proliferative response of A6-positive oval cells to DDCtreatment in WW45 Liv-cKO mice. (A) Five-week-old WW45 Liv-cKO orcontrol mice (n = 5) were fed a diet containing 0.1% DDC for the indicatedtimes, after which liver weight as a percentage of body weight was mea-sured. *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 micefed a 0.1% DDC diet for 4 weeks were subjected to H&E staining or to im-munohistochemical staining for A6, as indicated. Asterisks demarcate por-phyrin accumulation. (D) The area of A6 staining in liver sections wasquantitated with the use of ImageJ software for mutant and control micefed a 0.1% DDC diet for the indicated times. Data are expressed as a per-centage of thresholded areas. *P < 0.05 (n ≥ 3). (E) Oval cells isolated fromDDC-treated control or WW45 Liv-cKO mice were incubated in growth me-dium containing 10 μM BrdU for 5 h. The cells were then subjected to im-munofluorescence analysis (Upper) with antibodies to A6 (green) and toBrdU (red), and nuclei were stained with DAPI (blue). The percentage of A6-positive cells that were also positive for BrdU was determined for individualcolonies (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 werecultured in growth medium for 1 week and then subjected to immunoflu-orescence analysis (Upper) with antibodies to A6 (green) and to Ki67 (red);nuclei were stained with DAPI (blue). DIC images are shown in the top fourpanels. 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.)

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DiscussionWe previously showed that WW45 suppresses expansion of un-differentiated epithelial cells, including keratinocytes (5). However,the relevance of WW45 in progenitor cells of adults and in tumori-genesis remained unexamined. We have now generated mice withliver-specific ablation ofWW45 to address this issue. A recent studyhas underscored the importance of the Hippo pathway in control ofliver size and tumorigenesis via regulation of the proliferation ofdifferentiated hepatocytes (38). However, given that YAP, a down-stream effector of the Hippo pathway, contributes to expansion ofstemorprogenitor cells (1), it remainedpossible thatdysregulationofthe Hippo pathway in the liver might result in inappropriate expan-sionof liverprogenitor cells.Wehavenowfound that themammalianHippo–Sav pathway regulates homeostasis of progenitor (oval) cellsin the adult liver.WW45 Liv-cKO mice exhibited pronounced over-growth of the liver. Although this characteristic is similar to that ofmice with liver-specific knockout of MST1 and MST2, mammalianhomologs of Drosophila Hippo, the overproliferation of oval cells,not that of hepatocytes, contributes to suchovergrowth inWW45Liv-cKO mice. We thus found that the increase in liver size induced byexposure toDDCwas also enhanced in these animals comparedwiththat in controlmice. Finally,WW45Liv-cKOmice developed tumorswith a mixed (HCC/CC) phenotype, with such tumors being thought

to originate from transformed oval cells (13, 14, 16). These tumorsare thus distinct from the HCC, originating from aberrant pro-liferation of hepatocytes, that was observed in the MST1/2 condi-tional knockout mice (38). Our present findings therefore provideevidence that the mammalian Hippo–Sav pathway restricts the pro-liferation not only of epithelial progenitor cells during embryonicdevelopment but also of oval cells in the adult liver.YAP is frequently overexpressed inHCC (39), and liver-specific

overexpression of YAP results in enlargement of the liver and thedevelopment of liver tumors (2). YAP overexpression acting incooperation with cIAP1 and c-Myc in hepatic progenitor cells alsoleads to tumorigenesis (40). Consistent with the notion that theHippo pathway is the major suppressive regulator of YAP, abla-tion of WW45 in the liver also induces the formation of livertumors. We found that even liver tumors isolated from WW45+/−

mice, which retained the wild-type WW45 allele, manifested sub-stantial down-regulation of WW45 protein (Fig. S6C), suggestingthat the remainingwild-typeWW45 allelemay be silenced, possiblyas a result of promoter methylation. WW45 Liv-cKO mice alsoexhibited pronounced accumulation ofYAP in the liver.However,this accumulation ofYAPwas accompanied by that of otherHippopathway components and phosphorylated YAP. Nevertheless,a substantial amount of YAP was localized to nuclei in the muta-

Fig. 3. Deregulation of YAP as a result of liver-specific ablation ofWW45. (A) Liver lysates prepared from control (C) andWW45 Liv-cKO (cKO)mice either at theindicated ages (Left) or after maintenance on a 0.1% DDC diet for the indicated times (Right) were subjected to immunoblot analysis with antibodies to theindicated proteins, with GAPDH examined as a loading control. FL and NT, full-length and NH2-terminal fragment of MST1, respectively. (B) Nuclear (N) andcytosolic (C) fractionsof the liver of6-month-old (Upper) orDDC-treated (Lower) control andWW45 Liv-cKOmicewere subjected to immunoblotanalysis. LaminBandα-tubulinwereexaminedasnuclear and cytosolicmarker proteins, respectively. (C) Liver sections of control andWW45 Liv-cKOmice either at 6monthsof ageor 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 porphyrinaccumulation. (D) Excision PCR analysis of hepatocytes and anoval cell–enriched fraction (OEF) thatwere derived fromWTorWW45flox/flox (f/f)mice and infectedwith theAdeno-Cre virus (Upper). Arrow indicates the position of products (165 bp) derived from the excisedWW45 allele. Lysates of such infected cellswere alsosubjected to immunoblot analysis (Lower). Asterisks indicatenonspecific bands. (E)Oval cells isolated from the liver ofDDC-treated control orWW45Liv-cKOmicewere subjected to immunofluorescence staining for A6 (green) and YAP (red) as well as to stainingwith DAPI (blue). (F) Oval cells isolated from the liver of DDC-treated WT orWW45flox/flox mice were infected with Adeno-Cre and then subjected to immunofluorescence staining for A6 (green) and YAP (red) as well as tostainingwith DAPI (blue). (G) Oval cells isolated and infected as in Fwere incubated in growthmedium containing 10 μMBrdU for 5 h. The cells were then stainedwith 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 forindividual 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.)

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nt liver. Such localization was most evident in mutant oval cells,and these cells accordingly showed an increased proliferation in-dex. Furthermore, liver tumors in the mutant mice were enrichedin transformed oval cells. We therefore propose that the ablationofWW45 results in hyperactivation of YAP, preferentially in ovalcells, and thereby promotes liver tumorigenesis.The cleaved forms ofMST1andMST2were recently shown to be

required formaintenanceofhepatocyte quiescence in theadult liver(38). CleavedMST1/2, in contrast to the full-length proteins, cannotinteract withWW45as a result of loss of the SARAHdomain. Itwastherefore suggested that, whereas cleaved MST1/2 may be a keyregulator of YAP in differentiated hepatocytes, WW45 may playa minimal role. Although our data suggest that the cleaved form ofMST1may not be themajor formofMST1 in hepatocytes (Fig. 3A),they nevertheless indicate that WW45 indeed plays a minor role indifferentiated hepatocytes. Hepatocyte fractions lacking WW45thus showedonlymarginal, if any, up-regulationofYAP.WW45Liv-cKOmouse hepatocytes also showed a proliferative index similar tothat of control hepatocytes. An acute increase in liver size and tu-morigenesis due to proliferation of hepatocytes, similar to thoseapparent in both YAP transgenic mice (1, 2) and MST1/2 condi-tional knockout mice (38), were not observed in theWW45mutantliver. These results suggest that WW45 plays a minor role in main-tenance of hepatocyte quiescence.In contrast, we observed accumulation of YAP and other Hippo

pathway components in the oval cell fraction of theWW45-deficientliver. Accordingly, we found that ctgf and birc5, which were pre-viously shown to beup-regulated in the liver ofYAP transgenicmice(2) and MCF10A YAP-stable cell line (34), were specifically in-duced in oval cell–enriched fraction but not in isolated hepatocytes.

These findings suggest that WW45 in oval cells, unlike that inhepatocytes, plays a key role in the regulation ofYAP. Consistently,we found that the proliferation index of WW45-deficient oval cellswas increased both in vivo and in vitro.WW45 deficiency resulted inthe late development of mixed-type liver tumors indicative of ovalcell expansion. We therefore suggest that two distinct upstreamregulators of YAP operate in differentiated hepatocytes and in ovalcells. In hepatocytes, MST1/2 (Hippo) regulate YAP in a WW45(Sav)–independentmanner,whereas inoval cells,WW45 (Sav) is anessential regulator of YAP. The Hippo–Sav pathway is thus re-quired to restrict the proliferation of adult liver stem or progenitorcells. On the basis of our present data and our previous results withkeratinocytes (5), it is possible that WW45 functions as the majorsuppressorofYAPin stemorprogenitor cells ingeneral and therebyinhibits the inappropriate expansion of undifferentiated cells. Infact, recent reports suggested that the mammalian hippo pathwaymight be required to repress the oval cell activation (41, 42), sup-porting the hypothesis that Hippo signaling directly suppresses theproliferation of liver progenitor cells (oval cells).Although all Hippo pathway components were found to be up-

regulated in theWW45 Liv-cKO liver, we did not detect an increasein the abundance of the correspondingmRNAs. Such an increase inmRNA levels was apparent only after tumor development inWW45+/−mice, although the increase was not as pronounced as thatin protein levels (Fig. S6D). Regulation by stabilization of Hippopathway components therefore seems to be operative in normal livertissue, with feedback regulation of such components at the tran-scriptional level possibly operating only in the tumor environment.Further investigation of such regulatory mechanisms may provideimportant insight into homeostasis of Hippo signaling output.

Materials and MethodsGeneration of WW45 Liv-cKO Mice. Methods for generation of WW45 Liv-cKOmice are described in SI Materials and Methods.

DDC Feeding Protocol. For induction of oval cell proliferation, mice at ≈5weeks of age were fed a diet comprising standard chow supplemented with0.1% DDC (Sigma-Aldrich) (28).

Histologic Analysis. Liver tissue was fixed in 4% paraformaldehyde for prepa-ration of frozen sections and in 10% formalin for paraffin sections. DetailedprotocolsandallantibodiesforstainingaredescribedinSIMaterialsandMethods.

Isolation and Analysis of Hepatocyte and Oval Cell–Enriched Fractions. Hep-atocytes were prepared by a standard two-step perfusion protocol (43). Forisolation of oval cells, mice at 6–8 weeks of age were first fed a diet con-taining 0.1% DDC for 3 weeks, after which the liver was perfused in situ viathe hepatic portal vein as previously described (44). Methods for isolationand analysis of these cells are described in SI Materials and Methods.

Adeno-Cre Infection. Isolated primary hepatocytes and oval cells were in-cubated for 48 h with Adeno-Cre (kindly provided E.-J. Lee) at amultiplicity ofinfection of 100 pfu per cell (45). The cells were then exposed to fresh growthmedium and subjected to immunoblot or immunofluoresence analysis or toassay of BrdU incorporation.

Immunoblot Analysis and Subcellular Fractionation. Liver tissue as well as iso-latedhepatocyteoroval cell–enriched fractionswerehomogenized inPropreplysis buffer (iNtRON Biotechnology) containing protease and phophataseinhibitors. The lysateswere centrifugedat 15,000×g for 20minat 4 °C, and theresulting supernatantswere subjected to immunoblot analysis. Theantibodiesused for detection are described in SI Materials and Methods. Nuclear andcytosolic extracts were prepared with the use of NE-PER Nuclear and Cyto-plasmic Extraction Reagents (Pierce Biotechnology).

Statistical Analysis. Quantitative data are presented as means ± SD unlessindicated otherwise. Differences between means were evaluated by Stu-dent’s unpaired t test. P < 0.05 was considered statistically significant.

ACKNOWLEDGMENTS. We thank V. Factor (National Cancer Institute,Bethesda, MD) for kindly providing antibodies to A6; E.-J. Lee (Yonsei

Fig. 4. Mixed-type liver tumor development inWW45mutantmice. (A) Liver orhepatoma sections of WW45+/− mice at 8 or at 12 or 18 months of age, re-spectively, were subjected to H&E staining and to immunohistochemical stainingfor A6. (Insets) Small A6-positive oval cells in hepatic cords at a magnificationtwice that for the main panels. (B) Hepatoma sections fromWW45+/− orWW45Liv-cKOmice at 14 months of age were subjected to H&E staining and to immu-nohistochemical staining for A6. Strands or trabeculae of small A6-positive ovalcells were distributedwithin the hepatomas (a, a′, c, and c′). A6-positive oval cellswere also organized in glandlike structures within the tumors (b, b′, d, and d′).(C) Sections of normal liver or hepatoma tissue fromWW45+/+ orWW45+/−mice,respectively, were stained with antibodies to YAP. (D) Immunoblot analysis oftumors (T) at initiation, early, or advanced (Adv) stages and of correspondingnontumor (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-cKOmouse. (Scale bars, 200 μm in A; 100 μm in B; 50 μm in C.)

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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 LaboratoryProgram and the Korea National Cancer Center Program.

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