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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte-mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemochromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor–like weak inducer of apoptosis (TWEAK), is promitotic for LPCs; whether it acts directly is not known. In our murine choline-deficient, ethionine-supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor-inducible 14 (Fn14)] expression in the whole liver is massively upregulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE-induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2-week-CDE-treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin–positive cells. Stimulation of an Fn14-positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NFκB) activation and dose-dependent proliferation, which was diminished after targeting of the p50 NFκB subunit by RNA interference. Conclusion: TWEAK acts directly and stimulates LPC mitosis in an Fn14-dependent and NFκB-dependent fashion, and signaling via this pathway mediates the LPC response to CDE-induced injury and regeneration. (HEPATOLOGY 2010)

Hepatocyte-driven liver regeneration following acute injury is a highly orchestrated process in which remaining, healthy hepatocytes proliferate under the control of well-understood genetic, cellular, and metabolic networks.1 During chronic liver injury, however, when the functional liver mass is reduced and coincidentally hepatocyte proliferation is wholly or partly impaired, liver regeneration is facilitated by a compartment of dynamic, heterogeneous liver progenitor cells (LPCs),2 which in rodents are often called oval cells. LPCs are rarely detected in healthy tissue, but once activated, they proliferate and migrate into the parenchyma and differentiate into either cholangiocytes or hepatocytes. This process, unlike hepatocyte-mediated liver regeneration, is relatively poorly understood. Several factors mediating the LPC response to injury have been identified, including tumor necrosis factor (TNF), lymphotoxin β (LTβ), interferon γ (IFNγ), and transforming growth factor β, among others3-7; however, the mechanisms by which these mediators elicit their effects remain largely unknown. Thus, no definitive LPC growth factor has been described to date.

In this respect, the 2005 report by Jakubowski and coworkers8 was a milestone publication; it proposed a TNF family member, tumor necrosis factor–like weak inducer of apoptosis (TWEAK), as a direct LPC mitogen. Like most TNF superfamily ligands, TWEAK [also known as tumor necrosis factor (ligand) superfamily member 12, CD255, or previously APO3 ligand] is initially synthesized as a type II transmembrane protein that can be cleaved into a soluble cytokine.9 TWEAK, a noncovalent homotrimer, mediates its activity through binding to a 14-kDA type I transmembrane receptor termed fibroblast growth factor-inducible 14 (Fn14).10 Fn14 does not possess intrinsic protein kinase activity. Instead, it associates with tumor necrosis factor receptor–associated factor (TRAF) adaptor molecules and activates nuclear factor kappa light chain enhancer of activated B cells (NFκB) and the extracellular signal-regulated kinase and c-Jun N-terminal kinase mitogen-activated protein kinase pathways.11, 12 TWEAK regulates a diverse range of cellular processes, including proliferation, differentiation, migration, cell survival, and cell death, and has also been shown to act as a proangiogenic and proinflammatory factor.13

Although TWEAK is almost ubiquitously expressed in adult tissues, including the liver, its cognate receptor is barely detectable in hepatic tissue except during injury and repair. Feng et al.14 reported rapid induction of Fn14 expression during the early phases of regeneration following partial hepatectomy. The authors also investigated liver cancer–derived cell lines, and interestingly, Fn14 overexpression was mainly found in the poorly differentiated lines; this hinted at possible involvement in progenitor cells. This hypothesized relationship between LPCs and Fn14 signaling was strengthened by experiments described by Jakubowski and coworkers.8 In their study, it was demonstrated that hepatic TWEAK overexpression, after transgenic or adenoviral delivery, induces an LPC response; knockout (KO) of TWEAK/Fn14 inhibits the appearance of LPCs in an experimental model of oval cell proliferation. Furthermore, TWEAK directly induces proliferation of a biliary epithelial cell line with progenitor-like characteristics. LPCs consist of a heterogeneous cellular compartment, with different discrete subsets potentially being activated under various injury contexts. Thus, we wished to further explore the relationship between TWEAK and LPCs with a different in vivo model of hepatic injury and LPC proliferation developed in our laboratory.15 Additionally, we aimed to investigate the effects of TWEAK on LPC lines in vitro. We now demonstrate that TWEAK directly stimulates LPC mitosis in an Fn14-dependent and NFκB-dependent fashion and that this pathway plays a major role in the rapid growth phase of the LPC response to choline-deficient, ethionine-supplemented (CDE) diet–induced injury and regeneration.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animal Experimentation.

Fn14 KO mice and respective inbred wildtype (WT) animals were used as previously described.16 For all other experiments, C57BL/6 mice were obtained from ARC (Western Australia). Four-week-old, male mice were administered a CDE diet.15 Control animals received normal laboratory chow and drinking water. For TWEAK administration experiments (single injection), recombinant human tumor necrosis factor–like weak inducer of apoptosis (rhTWEAK; Biogen Idec, Inc., Cambridge, MA17) or vehicle (0.1% bovine serum albumin in normal saline) was injected intraperitoneally into 2-week-CDE-pretreated mice at a final dose of 0.02 μg/g of body weight. All procedures were performed in strict compliance with the guidelines set by the National Health and Medical Research Council of Australia and the University of Western Australia Animal Ethics Committee.

Primary LPC Isolates.

Livers of 2-week-CDE-treated mice were perfused, digested, and enriched for LPCs by discontinuous Percoll gradient centrifugation, as described previously.18

Cell Lines and Cell Culture.

The murine, bipotential LPC lines—bipotential murine oval liver (BMOL) and bipotential murine oval liver–tyrosine aminotransferase (BMOL-TAT)—were established from CDE-treated livers.18 Both lines were maintained as previously reported unless otherwise indicated. For proliferation experiments, serum and growth factor concentrations in the media were reduced to 2% fetal bovine serum (FBS), 15 ng/mL insulin-like growth factor II (IGF-II), 10 ng/mL epidermal growth factor (EGF), and 5 μg/mL insulin for three consecutive passages prior to use. Transfection was performed on 70% to 80% confluent cultures with either HiPerfect or SuperFect reagents (Qiagen, United States) according to the manufacturer's instructions.

Small Interfering RNA (siRNA) Knockdown.

Knockdown of NFκB p50 was performed under RNA interference (RNAi) conditions as previously described19 with commercial siRNAs (Qiagen) and a human/mouse siRNA starter kit (Qiagen).

Cell Proliferation.

Proliferation after exposure to rhTWEAK (Biogen Idec) was assessed in vitro in a medium containing 0.5% FBS, 15 ng/mL IGF-II, 10 ng/mL EGF, and 5 μg/mL insulin and was quantified with the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,20 the Cellscreen system,21 or an in vitro 5-bromo-2-deoxyuridine (BrdU) incorporation method (Calbiochem, United States). In situ, proliferation was evaluated via staining for Ki67 (Dako, United States), as previously described.19, 22

NFκB Assay.

The activity of NFκB in cell lines was measured with a green fluorescent protein (GFP) reporter transactivation assay (SABiosciences, United States) using the recommended methodology. After a 6-hour incubation step with the transfection complexes, cells were treated with rhTWEAK (Biogen Idec) for 24 hours, and GFP expression was quantitated fluorometrically and by fluorescence microscopy.

Fluorescence-Activated Cell Sorting (FACS) of Liver Leukocytes.

Leukocytes were prepared from phosphate-buffered saline–perfused livers as previously described.23 Cell suspensions were incubated with monoclonal antibodies [CD3-allophycocyanin, CD4-phycoerythrin (PE), CD8–fluorescein isothiocyanate, natural killer 1.1 (NK1.1)–PE, NK1.1–phycoerythrin cyanine 7 (PECy7), CD11b-PECy7, and CD11b-PE; all from BD Pharmingen, United States] at 1:50 dilutions for 30 minutes on ice, washed with an FACS wash, and sorted with a BD FACSVantage cell sorter (BD Biosciences). Cell duplets and propidium iodide–positive cells were excluded. The population of viable leukocytes was subdivided into CD3+ NK1.1 cells (T cells), CD3+NK1.1+ cells [natural killer T (NKT) cells], and CD3NK1.1+ cells (NK cells). T cells were further separated into CD4+ or CD8+ cells. NK, NKT, CD4+, and CD8+ T cells were collected in FBS. Macrophages were identified as CD3 NK1.1CD11bbright cells. About 10,000 cells per population (91%-99% pure) were collected for RNA extraction and real-time polymerase chain reaction analysis of TWEAK mRNA expression.

Gene Expression Analysis.

Total RNA was extracted with TRIzol (Invitrogen, Australia) according to the manufacturer's instructions and was quantified by spectrophotometry, and 1 μg of total RNA was reverse-transcribed with the ThermoScript system (Invitrogen). Quantitative polymerase chain reaction (qPCR) was performed with Quantitect (Qiagen) or the LightCycler 480 system (Roche Diagnostics, United States), and fluorescent output was monitored with the RG-3000 thermal cycler (Corbett Research, Australia) or LightCycler 480, respectively. The qPCR results were normalized against a housekeeping control gene (peptidylpropyl isomerase A, β-actin, or TATA box binding protein associated factor 4a) and expressed with respect to controls. Primer sequences are available upon request.

Immunofluorescence.

Immunofluorescence was performed on methanol/acetone-fixed liver cryosections, cytospins of BMOL cell lines, or primary LPC isolates18 according to a standard protocol with antibodies diluted in REAL antibody diluent (Dako, Denmark), and mounting was accomplished with the ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) for nuclear quantification. The primary antibodies were rat anti-A6 (1:100, Valentina Factor, Bethesda, MD), rat anti–cytokeratin 19 (anti-CK19; TROMA-3, 1:200, Rolf Kemler, Freiburg, Germany), rabbit anti–E-cadherin (24E10, 1:125, Cell Signaling, United States), rat anti-CD45 (Ly-5, 1:200, BD Pharmingen), rat anti-F4/80 (1:150, Ruth Ganss, Perth, Australia), rabbit anti–pan cytokeratin (anti-CKpan; 1:400, Dako), rat anti-Ki67 (TEC-3, 1:100, Dako), rabbit anti-NFκB p65 (C22B4, 1:30, Cell Signaling), rat anti-CD68 (FA-11, 1:200, Abcam, United States), mouse anti–alpha smooth muscle actin (anti-αSMA; ASM-1, 1:400, Chemicon, United States), and chimeric human Fc/mouse Fab anti-mouse Fn14 (chimP2D3, 2 μg/mL, or chimP4A8, 0.5 μg/mL, Biogen Idec). Primary antibodies were detected with goat anti-rabbit/rat Alexa Fluor 488, goat anti-rat/rabbit/mouse Alexa Fluor 594 (1:200, Invitrogen) or biotinylated goat anti-human (1:400, Vector Laboratories, United States), and streptavidin-PE (1:200, BD Biosciences) or streptavidin–Alexa Fluor 488 (1:800, Invitrogen). Stainings were quantified by either manual cell counting or with the AnalySIS Life Science Professional program (Olympus, Australia).

Sirius Red Staining.

Carnoy's solution–fixed and paraffin-embedded livers were sectioned, dewaxed, and rehydrated in distilled water before nuclear staining with hematoxylin, washing in distilled water, and 30-minute staining with 0.1% Sirius red in saturated aqueous picric acid.

Statistical Analyses.

Data are presented as means and standard errors of the mean (SEMs). Correlation was assessed by linear regression with PRISM (GraphPad Software, Inc., San Diego, CA). Statistical significance was assessed with one-way analysis of variance analysis and the Tukey posttest, Student t test, or Mann-Whitney U test (where applicable); this was facilitated with GraphPad InSat version 3.0b for Macintosh (GraphPad Software).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Fn14 and TWEAK Expression in the Liver.

Real-time polymerase chain reaction analysis of whole liver extracts showed low levels of TWEAK mRNA in all samples, with no significant difference between experimental groups (Fig. 1A). In both normal and CDE-injured livers, TWEAK was primarily expressed by natural killer (NK) cells and macrophages (Fig. 1B). Fn14 mRNA expression levels were substantially increased in CDE livers by approximately 6-, 17-, and 11-fold with 1, 2, and 3 weeks of CDE treatment, respectively (Fig. 1C). The correlation of Fn14 mRNA with LPC numbers (quantified by A6 staining and cell counting of 1- to 3-week CDE livers, as previously described)22 produced a significant (P < 0.0001) positive correlation with a coefficient of 0.9417 (Fig. 1D). Furthermore, primary nonparenchymal cell isolates from a 2-week CDE liver had proportions of cells positive for the LPC/biliary marker A6 (36%) and the LPC/epithelial cell marker E-cadherin (34%) similar to those for Fn14 (36%; not shown). Double immunofluorescence staining of these isolates illustrated coexpression of another accepted LPC/biliary marker, CKpan24 (Fig. 1E,G), and Fn14 (Fig. 1F,G) in all CKpan+ cells. To determine the phenotype of the small number of CKpan/Fn14+ cells, additional nonparenchymal cell markers were tested. Of these, only αSMA showed definite cellular colocalization with Fn14 in a small proportion of αSMA+ cells (Fig. 1H). Inflammatory cells (CD68+, CD45+) did not express Fn14 (Supporting Fig. 1).

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Figure 1. TWEAK and Fn14 expression in normal and regenerating livers. (A) qPCR analysis of the whole liver for TWEAK showed no significant change in the expression of TWEAK mRNA in CDE diet–fed mice versus control diet–fed controls (n = 6). (B) The isolation of liver leukocytes by flow cytometry and subsequent TWEAK qPCR showed that NK cells and macrophages were the major sources of TWEAK in both control liver and CDE liver (n = 2), with each replicate representing the combined RNA from two mice. (C) In contrast to TWEAK, whole liver Fn14 mRNA levels were massively increased in CDE-treated mice versus healthy animals (n = 3). (D) Linear regression analysis of the CDE liver on days 7, 14, and 21 showed that Fn14 mRNA levels correlated with the average number of A6+ LPCs present in the same liver (n = 3). Data represent means ± SEM, with statistical significance represented as *P < 0.05, **P < 0.01, and ***P < 0.001. The dashed line signifies mRNA levels of control diet–fed WT mice. (E) Percoll gradient isolates of primary LPCs from 14-day CDE liver contained clusters of cells positive for CKpan. (F,G) These clusters were almost always copositive for Fn14. (H) Double staining for Fn14 and αSMA showed both Fn14 (top panel) and Fn14+ (bottom panel) myofibroblasts.

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LPC Expansion Is Reduced in CDE-Fed Fn14 Null Mice.

The absence of Fn14 abrogated the linear expansion of LPCs in response to CDE diet–induced injury, and this led to a significant reduction in LPC numbers at 2 but not 3 weeks of treatment. Quantitation of sections stained with the biliary LPC markers A6 (Fig. 2A,D-I) and CK19 (Fig. 2B and Supporting Fig. 2) or the epithelial cell marker E-cadherin (Fig. 2C and Supporting Fig. 2), which was recently recognized as a marker for identification of LPCs in chronically injured livers,25 illustrated that this effect is independent of the marker used.

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Figure 2. LPC expansion is attenuated in Fn14-deficient animals. Fn14 KO or WT mice were subjected to a CDE diet to induce hepatocyte injury and stimulate the regenerative response of LPCs. After 2 weeks on the CDE diet, LPC numbers in WT livers were significantly increased versus control diet. In contrast, Fn14 KO mice exhibited only a modest enrichment of LPCs after 14 days of CDE treatment. Interestingly, however, the numbers of LPCs in the Fn14 KO livers normalized within 3 weeks on the diet, and they did not differ from the WT response at the 21-day time point. This pattern of LPC induction was evident, regardless of whether (A,D-I) A6 (A6 in red and DAPI for nuclear quantitation in blue), (B) CK19 (see Supporting Fig. 2), or (C) E-cadherin (see Supporting Fig. 2) was used to quantify the response. Data represent means ± SEM (n = 3-4). Statistical significance is represented as *P < 0.05. The dashed line signifies cell counts of control diet–fed WT mice.

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Inflammation and Fibrosis Correlate with Reduced LPC Expansion in Fn14 Null Mice.

Our group and others have previously described coregulation of inflammatory and fibrogenic responses with LPC proliferation in the CDE model.4, 22, 26, 27 Thus, we wished to determine whether the reduction of LPC proliferation observed in Fn14 null mice correlated with these other important regenerative pathologies. Numbers of both CD45+ leukocytes and F4/80+ macrophages were significantly reduced in Fn14 KO livers at 2 but not 3 weeks (Fig. 3A,B). In accordance, expression of the key inflammatory cytokines TNF (Fig. 3C), interleukin 6 (IL-6; Fig. 3D), IFNγ (Fig. 3E), and LTβ (Fig. 3F) also fell at 2 weeks. To assess fibrogenesis, livers were stained with Sirius red, and images were digitally quantified. Collagen deposition (Fig. 4A-C) and collagen-1 mRNA levels (Fig. 4E) correlated with LPC numbers in WT and Fn14 KO mice (Fig. 2), as observed in previous studies.4 CDE-induced mRNA expression of tissue inhibitor of metalloproteinases 1 (TIMP-1; Fig. 4F) and TIMP-2 (Fig. 4G), but not SRY (sex determining region Y)-box 9 (SOX-9; Fig. 4D), matrix metalloproteinase 2 (MMP-2; Fig. 4H), or MMP-9 (Fig. 4I), was reduced in Fn14-deficient animals at both 2 and 3 weeks.

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Figure 3. Inflammation is attenuated in Fn14-deficient animals. (A,B) Quantification of leukocytes (CD45+) or macrophages (F4/80+) in WT livers showed a modest (1.5- to 2-fold) enrichment in both cell types after 2 or 3 weeks of CDE feeding. This increase was attenuated in Fn14 KO animals at 14 days but rebounded by 21 days. Accordingly, mRNA expression of the proinflammatory cytokines (C) TNF, (D) IL-6, (E) IFNγ, and (F) LTβ, which increased in response to the CDE diet in WT mice, was suppressed in animals lacking Fn14, but again only at 2 weeks. Data represent means ± SEM (n = 3-4). Statistical significance is represented as *P < 0.05. The dashed line signifies cell counts or mRNA levels of control diet–fed WT mice.

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Figure 4. Fibrosis correlates with the LPC response in Fn14-deficient animals. (A,B) Sirius red staining and (C) digital quantification showed a spatiotemporal pattern of induction similar to that seen previously with LPC numbers. More specifically, after 2 weeks on the CDE diet, collagen deposition was clearly evident in the periportal areas, where LPC expansion also took place. (E) Associated with this was an increase in expression in collagen-1 mRNA. Additional fibrogenic mediator mRNA expression levels were also increased in the WT CDE liver compared to WT controls: (D) SOX-9, (F) TIMP-1, (G) TIMP-2, and (H) MMP-2 but not (I) MMP-9. Fn14-deficient mice showed a clear attenuation of fibrosis at 2 weeks as measured by (B,C) Sirius red, (E) collagen-1, and (F,G) suppression of TIMP expression. (C,E-G,I) By 3 weeks, this response appeared to have partially rebounded but remained mostly lower than that of WT mice. Data represent means ± SEM (n = 3-4). Statistical significance is represented as *P < 0.05 and **P < 0.01. The dashed line signifies mRNA levels of control diet–fed WT mice.

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TWEAK Is a Direct Mitogen to LPCs In Vitro.

The clonal LPC lines BMOL and BMOL-TAT18 were confirmed to be strongly positive for the TWEAK receptor Fn14 in the majority of cells (Fig. 5A). Treatment of BMOL and BMOL-TAT cells with rhTWEAK led to activation of the transcription factor NFκB, which was an expected outcome after stimulation of Fn14.11, 12 NFκB activation was dose-dependent, however, only at lower concentrations (Fig. 5B).

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Figure 5. LPCs are induced to proliferate by TWEAK. (A) The murine LPC lines BMOL and BMOL-TAT (isolated from a chronically injured CDE liver) were confirmed to be TWEAK-responsive by strong positivity for the receptor Fn14 in the vast majority of cells. (B) Treatment of BMOL cells with rhTWEAK induced activation of NFκB (as judged by a GFP reporter assay) in a dose-dependent fashion up to 10 ng/mL. (C,D) Accordingly, proliferation, as determined by either Cellscreen or MTT assay (not shown), was also dose-dependently induced by TWEAK, but again only at the lower end of the concentration ranges. Data represent means ± SEM (n = 4).

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Stimulation of growth factor- and serum-starved cells with rhTWEAK induced proliferation in a dose-dependent and time-dependent fashion, as determined by Cellscreen analysis (Fig. 5C) and confirmed by MTT assay (not shown). In agreement with the NFκB activation data, this effect was also dose-dependent only at doses up to 50 ng/mL (Fig. 5D).

TWEAK Stimulates LPC Proliferation In Vivo.

To address whether TWEAK can stimulate LPC proliferation in vivo, we administered rhTWEAK to mice with a preexisting LPC response induced by 2 weeks of CDE feeding and then measured proliferation in situ, 24 or 48 hours later, by staining for the proliferating cell marker Ki67. Hepatocyte proliferation did not differ between placebo and rhTWEAK-injected groups at either time point. In contrast, 24 hours after rhTWEAK administration, a 2-fold increase was observed in the number of Ki67-labeled nonparenchymal cells. By 48 hours post-rhTWEAK delivery, the proliferative wave had ended (Fig. 6A). Morphologically, many cells of the Ki67-labeled, nonparenchymal cell fraction had the appearance of LPCs and were located periportally. In accordance, double staining for CKpan24 and Ki67 illustrated that the majority of nonparenchymal cells stimulated by rhTWEAK injection were LPCs (Fig. 6B,C). Immunofluorescent staining for leukocytes revealed a time-dependent increase in CD45+ inflammatory cell numbers in rhTWEAK-injected animals compared to placebo-treated animals (Fig. 6D), and this is consistent with the previously demonstrated coregulation of the inflammatory response with LPC proliferation (Fig. 3).

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Figure 6. NFκB mediates the pro-proliferative effects of TWEAK on LPCs in vivo and in vitro. (A) The injection of recombinant TWEAK into mice with an existing LPC response, which was invoked by 2 weeks of CDE feeding, caused an increase in nonparenchymal (NP) cell proliferation 24 hours after injection but not 48 hours after injection. Hepatocytic cell proliferation (Heps) was not affected. (B,C) Double immunofluorescent staining showed that the majority of proliferating NP cells were CKpan+ LPCs. (D) CD45+ inflammatory cell numbers were significantly increased 48 hours after TWEAK treatment in comparison with placebo-injected controls. Staining of CDE liver sections 24 hours after TWEAK treatment for (E) the LPC/biliary marker A6 and (F) NFκB showed (G) numerous LPCs with active (nuclear) NFκB. (H) Knockdown of NFκB by RNAi reversed the pro-proliferative effects of TWEAK on LPC lines in vitro. Data represent means ± SEM (n = 4-6). Statistical significance is represented as *P < 0.05.

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NFκB Mediates TWEAK/Fn14-Induced LPC Proliferation.

Regenerating CDE livers showed the expected up-regulation of NFκB signaling in comparison with WT controls. Interestingly, numerous cells strongly positive for cytoplasmic and nuclear (active) NFκB were localized in periportal liver regions. Double staining of sections for A6 and NFκB demonstrated that many NFκB+ cells were represented by LPCs (Fig. 6E-G); this suggests that this transcription factor mediates the pro-proliferative effects of TWEAK in the CDE model. To test this hypothesis, RNAi was used to knock down the p50 NFκB subunit in the LPC lines BMOL and BMOL-TAT (which were isolated from a CDE-treated liver) before measurement of the mitotic S phase by BrdU incorporation. Transfection of p50 siRNA reduced TWEAK-induced LPC proliferation in a dose-dependent fashion. Dose-dependent proliferation of LPCs following TWEAK stimulation was not affected by transfection of a scrambled control siRNA vector (Fig. 6H).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Once considered by many to be little more than a histological artifact, adult LPCs (oval cells) are now the subject of much attention because of their potential use as vectors or targets for cell or drug-based therapy28 and their clear association with chronic hepatic disease progression.6, 19, 29 With respect to the former, it is interesting to note that despite considerable efforts, no LPC-specific growth or differentiation factors have been identified. The characterization of TWEAK as a mitogen for LPCs not only is important for our understanding of how regenerative responses are mediated in chronic damage but also draws us one step closer to achieving selective modulation of LPC behavior for the purposes of cell therapy and regenerative medicine.

The study by Jakubowski et al.8 showed definitively that the TWEAK ligand and its cognate receptor Fn14 mediate the behavior of at least a subset of LPCs. This was demonstrated by several approaches, including (1) gene-targeted mice, which showed a reduced presence of A6-expressing cells in an experimental model of ductular reaction, and (2) increased numbers of LPCs in transgenic mice, which overexpressed TWEAK. LPCs are recognized as being highly heterogeneous and dynamic, and several previous studies from our laboratory have illustrated selective modulation of discrete subpopulations of LPCs by cytokines.3, 30, 31 Because the LPC compartment induced in various liver injury models may be different, we aimed to further explore the role of the TWEAK pathway in an additional in vivo model of LPC expansion and to study its effects directly on LPC lines in vitro.

Using the CDE model of hepatocellular injury and regeneration, which our laboratory pioneered for use in mice,15 we now show that TWEAK signaling via its receptor Fn14 is essential for the early expansion of LPCs in response to CDE-induced liver injury. In livers from CDE diet–fed mice, LPCs proliferate outward from periportal regions, not in ducts or pseudoducts, but as streams of cells. We have extensively studied the dynamics of this response and shown that the LPC proliferation in this model has two phases: linear growth and maintenance. Linear growth begins between days 3 and 5 and is completed around days 12 to 16. During this phase, LPCs have a doubling time of approximately 18 hours, a cycling time typical of a stem or progenitor cell.32 At the end of the linear growth phase, LPC numbers plateau, and only small numbers of LPCs are seen dividing (B. Knight, unpublished observations, 2009). Thus, it is interesting that Fn14 KO mice displayed significantly fewer LPCs on day 14 but not day 21; this suggests that TWEAK is important in mediating the rapid expansion of the LPC pool immediately after the onset of liver damage and is less significant in affecting their slow turnover in the latter phase of the diet response. One interpretation is that the linear LPC expansion phase is delayed by the lack of TWEAK signaling, and other pathways compensate subsequently. Many cytokines orchestrate the LPC response, and it is therefore also not surprising that a deficiency of a single cytokine inhibits but does not fully block LPC proliferation, as has been shown for CDE-fed mice deficient for TNFR1, TNF/LTα, IFNγ, or LTβ.3, 5, 6 In this respect, it is important to note that Jakubowski et al.8 also used the 14-day time point to examine the response of Fn14 KO mice to 3,5-diethoxycarbonyl-1,4-dihydrocollidine–induced injury. Preliminary data detailing the dynamics of the LPC response in this model suggest that rapid growth also occurs between approximately days 5 and 16 (B. Knight, unpublished observations, 2009), with peak Ki67 labeling of LPCs on day 12 (Boulter et al., unpublished data, 2009). In our in vivo studies, we have not detected an increase in TWEAK mRNA expression in response to injury. Expression of TWEAK in CDE cell isolates was predominantly in NK cells and macrophages, which are known to associate physically with proliferating LPCs in CDE and other models.33 In contrast, Fn14 expression was massively up-regulated in CDE samples and correlated positively with LPC numbers. In primary cell isolates from a CDE liver, all cells positive for the LPC/biliary marker CKpan coexpressed Fn14. We initially suspected that the small proportion of CKpan/Fn14+ cells were LPCs reflecting a more hepatocytic gene expression profile and thus negative for the biliary antigen CKpan. However, double staining for other nonparenchymal cell markers with Fn14 yielded an unexpected result: the myofibroblast marker αSMA colocalized with Fn14 in a subset of cells. The implication of this, at this time, is not entirely clear as a lineage relationship between LPCs and hepatic stellate cells has been proposed.34 Regardless, we propose that a paracrine TWEAK-Fn14 signaling system operates in LPC-mediated liver regeneration, in which localized secretion of the TWEAK ligand by NK cells and macrophages regulates LPC behavior by binding and activating Fn14 on LPCs.

Another novel finding from our study is the coregulation of inflammation and collagen deposition with LPC responses in TWEAK signaling–deficient mice. Previous studies have suggested that these responses go hand in hand,4, 22, 35, 36 and again in the present study, we have robustly demonstrated this correlation. We previously hypothesized that inflammatory infiltration precedes the progenitor cell response in CDE-fed mice,32 and we more recently proposed that initiation of LPC proliferation stimulates activation of hepatic stellate cells and thus matrix remodeling.4 Interestingly, Van Hul et al.26 disagree with our proposed sequence; they have suggested that the fibrosis response occurs before LPC expansion. In CDE-fed Fn14 null mice, the hepatic inflammatory response was delayed, and this is consistent with reduced inflammation in cardiotoxin-treated Fn14 null animals in a model of skeletal muscle regeneration.16 Collagen deposition correlated with the LPC response, with a reduction evident at 2 but not 3 weeks of CDE feeding. If TWEAK is a direct and selective mitogen for LPCs, this implies that fibrosis follows the LPC response and not the other way around. However, as inflammation was coregulated in the Fn14 KO mice, it is not possible from this alone to resolve the relative sequence of the two events.

To demonstrate that TWEAK is a definitive LPC mitogen, development of an in vitro system was essential. In this respect, it is important to note that Jakubowski et al.8 used a well-differentiated biliary epithelial cell line. We have previously established immortalized LPC lines from WT or transgenic CDE-treated mouse livers and characterized as bipotential murine oval liver (BMOL) cells.18 As early as 24 hours after TWEAK exposure, growth of the BMOL lines was notably stimulated; this continued for up to 6 days with no plateau apparent. Proliferation was dose-dependent up to 50 ng/mL rhTWEAK; at 100 ng/mL, however, growth was slightly reduced, as was NFκB activation. RNAi experiments have demonstrated the central role of NFκB in mediating Fn14 signaling. When TWEAK binds Fn14, it recruits adaptor molecule TRAF2 (or TRAF5), which activates the NFκB signaling complex and thus results in a long-lasting prosurvival, pro-proliferative response.12 In accordance with an important role for NFκB in mediating LPC expansion, we found numerous cells expressing both inactive (cytoplasmic) and active (nuclear) NFκB in CDE-treated livers. Considering all this, we suggest that TWEAK signaling, via Fn14 to NFκB, mediates the rapid growth of LPCs in the first 14 days of CDE feeding. These findings are important in the context of manipulating cells for possible therapeutic applications and for future experiments investigating the pro-proliferative and anti-proliferative effects of growth factors and cytokines on LPCs in vitro and in vivo.

In our model, we propose that local production of TWEAK by NK cells and macrophages mediates the expansion of LPCs in chronic injury models, but it is significant that we can show that delivery of exogenous rhTWEAK induces proliferation of periportal CKpan-expressing nonparenchymal cells showing LPC morphology. We detected a mean doubling in proliferating nonparenchymal cells 24 hours after TWEAK administration. By 48 hours, numbers had returned to the baseline. The majority of Ki67+ nonparenchymal cells (∼60%) were LPCs (as judged by their CKpan positivity24). It should be emphasized, however, that this is a conservative estimate, as we have shown previously that in the CDE model, only a subset of LPCs is A6/CKpan-positive.18 It is therefore likely that some of the Ki67+/CK cells are nonbiliary LPCs. This suggests that it might be possible to augment LPC-mediated liver regeneration with TWEAK, and there are implications for its use in patients with chronic liver disease. However, such studies need to proceed with care, given our findings that a subset of myofibroblasts is also Fn14+ and thus may also respond to TWEAK stimulation.

In summary, demonstrating definitively that TWEAK is a mitogen for LPCs in vitro and in vivo, this study has confirmed and extended the previous work of Jakubowski et al.8 On the basis of concurrent analysis of isolated and in situ LPCs, we conclude that the TWEAK signaling system is activated in the chronically injured liver by up-regulated expression of the Fn14 receptor on existing and newly produced LPCs. TWEAK protein is produced and secreted by neighboring NK cells and macrophages and, once bound to Fn14, activates NFκB and, in turn, expression of numerous cell cycle regulatory factors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors are grateful to Ben Dwyer for technical assistance and to Dr. Valentina Factor, Associate Professor Ruth Ganss, and Professor Rolf Kemler for providing antibodies for this study.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_23663_sm_SuppFigure1.tif11284KSupporting Information Figure 1. Fn14 expression in primary liver cell isolates. In primary cell isolates from 2-week CDE liver, neither CD68+ macrophages (A-C) nor CD45+ leukocytes in general (D-F) stained for the TWEAK receptor Fn14. However several cells double positive for αSMA and Fn14 were identified (G-I), suggesting that some of the CKpan-/Fn14+ cells present in primary cell isolates could be represented by myofibroblastic hepatic stellate cells.
HEP_23663_sm_SuppFigure2.tif15166KSupporting Information Figure 2. LPC expansion is attenuated in Fn14-deficient animals. Fn14 KO or WT mice were subjected to a CDE diet to induce chronic liver injury. In healthy WT liver, only very few cells of biliary origin stained for CK19 (A, D; CK19 in red, DAPI for nuclear quantitation in blue). After two-week CDE treatment, significantly more CK19+ LPCs proliferated in WT than in Fn14 KO mice (B, C). However, numbers of LPCs in Fn14 KO livers normalised by three weeks on the diet, not differing from the WT response at the 21-day time point (E, F). Quantitation of non-parenchymal E-cadherin+ cells showed the same staining pattern as seen for CK19+ cells in all experimental groups (G-L).

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