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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Am J Transplant. 2012 Jun 13;12(8):2052–2061. doi: 10.1111/j.1600-6143.2012.04069.x

Amphiregulin Stimulates Liver Regeneration after Small-for-Size Mouse Liver Transplantation

Q Liu 1,5, H Rehman 1, Y Krishnasamy 1, K Haque 1, RG Schnellmann 1,4, JJ Lemasters 1,2,3, Z Zhong 1,3
PMCID: PMC3409348  NIHMSID: NIHMS365859  PMID: 22694592

Abstract

This study investigated whether amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), improves liver regeneration after small-for-size liver transplantation. Livers of male C57BL/6 mice were reduced to ~50% and ~30% of original sizes and transplanted. After transplantation, AR and AR mRNA increased in 50%- but not in 30%-grafts. 5-Bromodeoxyuridine (BrdU) labeling, proliferating cell nuclear antigen (PCNA) expression, and mitotic index increased substantially in 50%- but not 30%-grafts. Hyperbilirubinemia and hypoalbuminemia occurred and survival decreased after transplantation of 30%- but not 50%-grafts. AR neutralizing antibody blunted regeneration in 50%-grafts whereas AR injection (5 μg/mouse, iv) stimulated liver regeneration, improved liver function and increased survival after transplantation of 30%-grafts. Phosphorylation of EGFR and its downstream signaling molecules Akt, mTOR, p70S6K, ERK and JNK increased markedly in 50%- but not 30%-grafts. AR stimulated EGFR phosphorylation and its downstream signaling pathways. EGFR inhibitor PD153035 suppressed regeneration of 50%-grafts and largely abrogated stimulation of regeneration of 30%-grafts by AR. AR also increased cyclin D1 and cyclin E expression in 30%-grafts. Together, liver regeneration is suppressed in small-for-size grafts, as least in part, due to decreased AR formation. AR supplementation could be a promising therapy to stimulate regeneration of partial liver grafts.

Keywords: growth factor, liver graft survival, liver regeneration, liver transplantation, living donor transplantation, small-for-size syndrome

INTRODUCTION

Partial liver transplantation (PLT) has been practiced extensively in recent years to alleviate the severe shortage of donor livers (1–3). A critical factor for survival and rapid recovery of graft function after PLT is the size of grafts to be transplanted (1;4). Small-for-size syndrome usually occurs when the relative graft volume is less than 30–40% of the standard liver volume, leading to slow/no recovery of liver function and ultimately graft failure (1;4;5). The mechanisms of small-for-size graft failure remain unclear. Previous studies showed that liver regeneration is inhibited in small-for-size grafts, which was associated with compromised graft function (6–11).

Liver regeneration is regulated by a variety of genes, transcription factors, cytokines, and growth factors (12;13). Two receptor-ligand and growth factor signaling systems appear to be mainly involved in liver regeneration: the epidermal growth factor receptor (EGFR) and its relatively large family of ligands and coreceptors as well as hepatic growth factor (HGF) and its receptor (Met) (14;15). Other signaling pathways such as Notch/Jagged and c-kit as well as energy status, nutritional factors, hormones and free radicals also affect liver regeneration (10;13;16–18). Tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), and HGF, the major cytokines and growth factor that stimulate liver regeneration, increased after transplantation of small-for-size liver grafts (6;9). Therefore, the suppression of regeneration in small-for-size grafts appears not due to lack of these growth factors and cytokines. Here, we sought to determine other growth factors/cytokines that might contribute to suppression of regeneration of small-for-size grafts.

Amphiregulin (AR) is a primary mitogen for hepatocytes, acts as an early trigger of liver regeneration, and genetic deletion of AR leads to decreased liver regeneration (14;19). AR is synthesized as a transmembrane precursor which is proteolytically processed by ADAM metallopeptidase domain 17 (ADAM17), forming the mature form which binds to the epidermal growth factor receptor (EGFR) (20). Binding of ligands to EGFR leads to EGFR dimerization, intrinsic protein tyrosine kinase activation, tyrosine autophosphorylation, and subsequent activation of several intracellular signaling pathways, such as phosphatidyl inositide 3 kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways and the Ras-Raf mitogen activated protein kinase (21;22). A previous study showed that inhibitors of EGFR, MEK-1, PI3K and JNK blocked AR-induced DNA synthesis in cultured hepatocytes, whereas p38 MAPK inhibition had no effect (19). Whether AR expression is altered in small-for-size liver grafts remains unclear. Therefore, this study also investigated the role of AR in regeneration of small-for-size livers and the effects of AR supplementation on the outcomes of PLT.

MATERIALS AND METHODS

Animals and Partial Liver Transplantation

Orthotopic liver transplantation (LT) was performed in male C57BL/6 mice (10–11 weeks) under isofluorane anesthesia (23). The left lateral lobe, the anterior and posterior caudate lobes plus the median lobe or the left portion of the median lobe were removed after ligation with 5-0 silk suture resulting reduction of liver mass to ~30% (thirty percent-size grafts, TSG) and ~50% (half-size grafts, HSG), respectively. Full-size grafts (FSG), HSG and TSG were explanted and stored in lactated Ringer’s solution for 2 h. The transplantation procedures are described in “Supplements.”

AR (5 μg/mouse, iv, R&D Biosciences, Minneapolis, MN), AR neutralizing antibody (100 μg/mouse, iv; R&D Biosciences) and EGFR inhibitor PD153035 (30 mg/kg, ip; R&D Biosciences), or an equal volume of vehicles (normal saline for AR and AR antibody; DMSO for PD153035) was injected immediately after implantation. Survival was monitored for 7 days following surgery.

Clinical Chemistry, Histology and Immunohistochemistry

Blood and livers were harvested 5 and 48 h after transplantation. Serum alanine aminotransferase, total bilirubin and albumin were measured as described elsewhere (16). Histology was examined after hematoxylin-eosin (H&E) staining (24). Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (25). BrdU incorporation in liver sections was determined immunohistochemically and quantified as described previously (16). Mitotic cells were counted in a blinded manner in 10 random fields in H&E stained liver sections using a 40x objective lens.

Quantitative Real-Time PCR and Immunoblotting

mRNAs of AR, TNFα, IL-6, and HGF were determined using qPCR. Proteins of interest were detected by immunoblotting. These methods are described in “Supplements” (25).

Statistical Analysis

Data shown are mean±S.E.M. Groups were compared using the Kaplan-Meier test and ANOVA plus Student-Newman-Keuls as appropriate. Numbers of animals were 8 to 10 per group in the survival experiment and 4 per group for all other parameters, as indicated in figure legends. Differences were considered significant at p<0.05.

RESULTS

AR Expression is Inhibited in Small-for-Size Liver Grafts

Previous studies showed that AR peaked at 5–6 h after partial hepatectomy (PHX)(19). Therefore, we investigated changes in AR at 5 h after LT. AR was expressed at low levels in sham-operated livers and FSG but increased ~14-fold in HSG (Fig. 1A and B). By contrast, AR did not increase in TSG (Fig. 1A and B). AR mRNA did not change in FSG, increased 2.2-fold in HSG and decreased in TSG, suggesting increased synthesis of AR in HSG but not in TSG (Fig. 1C).

Fig. 1. Decreased Amphiregulin Formation in Small-for-Size Liver Grafts after Transplantation.

Fig. 1

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Full-size and reduced-size mouse livers were transplanted, as described in “METHODS.” Liver grafts were harvested 5 h after implantation. Amphiregulin (AR) and ADAM17 were detected by immunoblotting, and representative gels of 4 livers per group were shown (A). Panel B plots AR/actin ratios determined by densitometry. Panel D plots the values of ADAM17/actin. AR mRNA was determined by real-time PCR (C). Values are mean ± SEM (n = 4/group). a, p<0.05 vs. sham-operation; b, p<0.05 vs. FSG (100%); c, p<0.05 vs. HSG (50%).

ADAM17 cleaves and releases AR from the cell surface so that it can interact with its receptor (20). ADAM17 was expressed at low levels in sham-operated livers (Fig. 1A and D). After transplantation, ADAM17 did not change in FSG but increased 1.8-fold and 3.2-fold in HSG and TSG, respectively. Therefore, decreased liver regeneration is not due to inhibition of AR activation.

HB-EGF, another ligand of the EGFR, was barely detectable in sham-operated livers, FSG and TSG (Fig. 1A). HB-EGF increased very modestly in HSG (Fig. 1A). EGF was not detectable in sham-operated livers and FSG (Fig. 1A). EGF increased very modestly after HSG and TSG transplantation (Fig. 1A). TGFα was barely detectable in sham-operated livers and FSG (Fig. 1A). TGFα markedly and equally increased in HSG and TSG (Fig. 1A). Together, among the EGFR ligands we detected, only the alterations of AR were consistent with the regenerative responses after PLT (described below).

We also examined HGF, TNFα and IL-6 mRNAs after PLT (Fig. 2). HGF mRNA did not increase in FSG but increased 1.9-fold and 2.8-fold in HSG and TSG, respectively. Similar trends were observed for TNFα and IL-6 mRNAs, consistent with previous observations (6).

Fig. 2. Increased Hepatic Growth Factor, Tumor Necrosis Factor-α and Interleukin-6 Formation in Small-for-Size Liver Grafts after Transplantation.

Fig. 2

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Full-size and reduced-size mouse livers were transplanted, as described in “METHODS.” Liver grafts were harvested 5 h after implantation. HGF (A), TNFα (B) and IL-6 (C) mRNAs were detected by real-time PCR. A.U., arbitrary unit. Values are mean ± SEM (n = 4/group). a, p<0.05 vs. sham-operation; b, p<0.05 vs. FSG (100%); c, p<0.05 vs. HSG (50%).

Liver Regeneration is Inhibited in Small-for-Size Liver Grafts: Reversal by AR Supplementation

Since AR expression increased in HSG but was inhibited in TSG (Fig. 1), we investigated whether AR neutralization would suppress and AR supplementation improve regeneration of small-for-size liver grafts. BrdU labeling, which indicates DNA synthesis in cell cycle, was barely detectable 48 h after sham-operation and in FSG (Fig. 3A and B, Fig. 4A). BrdU labeling increased to ~23% in HSG. AR neutralizing antibody decreased BrdU incorporation to ~10% (a 57% decrease) in HSG (Fig. 4A). BrdU labeling was only 1% in TSG. Treatment of recipients with AR (5 μg/mouse), a dosage which caused maximal liver regeneration in mice in preliminary experiments, increased BrdU labeling in TSG to ~23% (Figs. 3D and E and Fig. 4A).

Fig. 3. 5-Bromo-2′-Deoxyuridine Incorporation is Suppressed after Transplantation of Small-for-Size Liver Grafts: Reversal by AR Treatment.

Fig. 3

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Full-size and reduced-size mouse livers were transplanted. Some TSG recipients were treated with AR (5 μg/mouse, iv) immediately after implantation. Liver grafts were harvested 48 h later. BrdU incorporation was detected immunohistologically and representative images are shown (n = 4/group). Panels are: A, sham-operation; B, FSG (100%); C, HSG (50%); D, TSG (30%); E, TSG treated with AR. Bar is 50 μm.

Fig. 4. Liver Regeneration is Suppressed after Transplantation of Small-for-Size Liver Grafts: Reversal by AR Treatment.

Fig. 4

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Full-size and reduced-size mouse livers were transplanted. Some HSG recipients were injected with AR neutralizing antibody (α-AR, 100 μg/mouse, iv), and some TSG recipients were treated with AR (5 μg/mouse, iv) immediately after implantation. Liver grafts were harvested 48 h later. BrdU-positive and -negative cells in 10 random fields per slide were counted under microscope with a 40x objective lens. The percentage of BrdU-labeled hepatocytes is plotted in A. PCNA was determined by immunoblotting, representative gels of 4 livers/group are shown in B. Mitotic hepatocytes were counted in 10 random fields per H&E stained liver section and plotted in C. a, p<0.05 vs. sham-operation; b, p<0.05 vs. FSG (100%); c, p<0.05 vs. HSG (50%), d, p<0.05 vs. HSG (50%) with AR neutralizing antibody treatment; e, p<0.05 vs. TSG (30%).

PCNA, which acts as a processivity factor for DNA polymerase δ (26), was used as another indicator of cell proliferation. PCNA was barely detectable in sham-operated livers and did not increase in FSG (Fig. 4B). PCNA expression increased markedly in HSG at 48 h, which was blunted by AR neutralizing antibody (Fig. 4B). By contrast, PCNA increased only slightly in TSG. AR recovered PCNA expression in TSG to the level of HSG.

Mitotic cells were minimal in sham-operated livers and FSG. After transplantation, mitotic index increased to 8% in HSG (Fig. 4C), which was blunted by AR neutralization. Mitotic cells did not increase in TSG, but AR restored mitosis of hepatocytes in TSG to 75% of the level of HSG (Figs. 4C). Together, these data indicate that liver regeneration was suppressed in TSG and AR supplementation reversed this suppression.

AR Supplementation Improves Function and Survival of Small-for-Size Liver Grafts

Serum total bilirubin was 0.04 mg/dL in sham-operated mice and was not significantly altered at 48 h after transplantation of FSG and HSG. Total bilirubin increased to 3 mg/dL after TSG transplantation, indicating poor graft function (Fig. 5A). Supplementation of AR blunted hyperbilirubinemia by 63%. Serum albumin was ~4 g/dL which was not significantly altered after FSG or HSG transplantation (Fig. 5B). Albumin decreased to 0.9 g/dL after TSG transplantation, possibly due to leakage of albumin from the vascular system, urinary excretion, increased degradation by activated proteases and/or decreased albumin synthesis by dysfunctioning TSG. AR supplementation increased serum albumin to 2.5 g/dL (Fig. 5B).

Fig. 5. Decreased Liver Function and Survival after Transplantation of Small-for-Size Liver Grafts: Reversal by AR.

Fig. 5

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Blood was collected 48 h after transplantation. Serum total bilirubin (A) and albumin (B) were determined (n = 4/group). a, p<0.05 vs. sham-operation; b, p<0.05 vs. FSG (100%); c, p<0.05 vs. HSG (50%), d, p<0.05 vs. TSG (30%). Mice were observed 7 days post-operatively for survival (C). Group sizes were 10 each in the FSG, HSG and TSG group, respectively, and 8 in the TSG+AR group. P<0.05 by the Kaplan-Meier test.

All mice survived after FSG and HSG transplantation (Fig. 5C). In contrast, only 10% of mice survived 7 days after TSG transplantation (Fig. 5C). Importantly, AR increased survival of mice with TSG to 75% (Fig. 5C). Together, these data show that AR improves recovery of liver functions and survival of small-for-size liver grafts.

AR Supplementation Does not Prevent Injury in Small-for-Size Liver Grafts

At 48 h after LT, necrosis was minimal in FSG and HSG (Suppl. Fig. 1). In particular, small focal necrosis was observed in TSG with and without AR treatment to approximately the same extent. One week after LT, TSG treated with AR showed mild biliary proliferation but hepatocyte morphology largely recovered to normal.

Apoptosis was evaluated by TUNEL staining. TUNEL-positive cells were 0.13% in sham-operated livers, which was not significantly altered in FSG and HSG. TUNEL-positive cells increased mildly to 0.73% in TSG (Suppl. Fig. 2A). AR did not decrease apoptosis in TSG.

Serum ALT increased from 37 to 100, 256, and 911 U/L at 48 h after transplantation of FSG, HSG and TSG, respectively. AR did not decrease ALT release from TSG (Suppl. Fig. 2B).

Decreased EGFR Activation in Small-for-Size Liver Grafts: Reversal by AR Supplementation

Binding of ligands to EGFR leads to its activation (21;22). Phospho-EGFR existed at low levels in sham-operated livers and FSG (Figs. 6A and B). At 5 h after transplantation, phospho-EGFR increased 4.8-fold in HSG but remained at lower levels in TSG. AR supplementation increased phospho-EGFR in TSG to a level that was equal to HSG (Figs. 6A and B). EGFR expression was not statistically different between sham-operation and all other groups after transplantation (Fig. 6A).

Fig. 6. Decreased EGFR Activation in Small-for-Size Liver Grafts: Reversal by AR Treatment.

Fig. 6

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Livers were collected 5 h after transplantation. EGFR, phospho-EGFR and actin were detected by immunoblotting, representative gels are shown in A (n = 4/group), and intensities of phospho-EGFR signals quantified by densitometry and standardized by actin are shown in B. a, p<0.05 vs. sham-operation; b, p<0.05 vs. FSG (100%); c, p<0.05 vs. HSG (50%), d, p<0.05 vs. TSG (30%) (n = 4/group). Some HSG recipients and TSG recipients with AR treatment were injected with the EGFR inhibitor PD153035 (30 mg/kg, ip) immediately after implantation. Percentage reduction of BrdU incorporation (BrdU) and mitotic index (MI) by PD153035 is shown in C (n=4 per group). **, p<0.01 vs HSG; ##, p<0.01 vs TSG recipients with AR treatment.

To assess the importance of EGFR in hepatic regeneration after PLT, recipient mice were treated with PD153035, an EGFR inhibitor (19), immediately after implantation. PD153035 treatment decreased BrdU incorporation and mitotic index in HSG by 61% and 66%, respectively (Fig. 6C). In TSG treated with AR, PD153035 blunted increases in BrdU incorporation and mitotic index by 96% and 97%, respectively (Fig. 6C).

Alterations of EGFR Downstream Signaling Pathways after PLT

EGFR leads to activation of several downstream signaling pathways. Therefore, we investigated which pathway(s) mediated the effects of AR in TSG. Akt was expressed equally in sham-operated livers and all liver grafts (Fig. 7). Phospho-Akt existed at low levels in sham-operated livers and FSG but increased 5.9-fold in HSG 5 h after transplantation, indicating activation of the PI3K pathway (Fig. 7 and Suppl. Fig. 3A). In contrast, phospho-Akt was not significantly increased in TSG. AR supplementation completely recovered Akt phosphorylation in TSG (Fig. 7 and Suppl. Fig. 3A). mTOR was expressed equally in sham-operated livers and all liver grafts (Fig. 7). Phospho-mTOR was barely detectable in sham-operated livers and FSG but increased 14-fold in HSG (Fig. 7 and Suppl. Fig. 3B). mTOR activation did not increase in TSG. After AR treatment in TSG, mTOR activation recovered to the levels of HSG (Fig. 7 and Suppl. Fig. 3B). Phosphorylation of p70S6K, a kinase downstream of mTOR, exhibited the same trend (Fig. 7 and Suppl. Fig. 3C).

Fig. 7. Alterations of EGFR Downstream Signaling Molecules after Liver Transplantation: Role of Amphiregulin.

Fig. 7

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Livers were collected 5 h after transplantation. phospho-Akt, Akt, phospho-mTOR, mTOR, phospho-p70S6 kinase (p-p70S6K), ERK, phospho-ERK, JNK, phospho-JNK and actin were detected by immunoblotting. Representative gels are shown (n = 4/group).

Expression of ERK1/2 was not different among groups 5 h after sham-operation or transplantation (Fig. 7). Phospho-ERK1/2 was not altered in FSG compared to sham-operated livers but increased 7-fold in HSG (Fig. 7 and Suppl. Fig. 3D). Phospho-ERK1/2 increased only 2.4-fold in TSG. AR treatment increased both ERK1 and ERK2 phosphorylation. In AR-treated TSG, phospho-ERK1/2 increased 8-fold.

At 5 h after transplantation, JNK1/2 expression was not different between groups (Fig. 7). Phospho-JNK1 was undetectable and phospho-JNK2 was expressed in very low levels in sham-operated livers (Fig. 7). Phospho-JNK1/2 did not change in FSG (Fig. 7). Phospho-JNK2 increased substantially whereas phospho-JNK1 increased only slightly in HSG (Fig. 7 and Suppl. Fig. 3E). In contrast, both phospho-JNK1 and 2 remained at low levels in TSG. With AR supplementation, both phospho-JNK1 and 2 in TSG were restored to or above the levels of HSG (Fig. 7 and Suppl. Fig. 3E).

Cyclin Expression Inhibition in Small-for-Size Liver Grafts: Reversal by AR Treatment

Cyclins control the progression of cells through the cell cycle (27). CyD1, an important cell cycle regulator and an important target of the JNK/c-Jun, ERK and mTOR pathways in driving regeneration (28–30), was barely detectable in sham-operated livers and FSG (Fig. 8A) but increased 18-fold in HSG (Fig. 8A and B). In contrast, CyD1 expression did not increase in TSG (Fig. 8A & B). In TSG treated with AR, however, CyD1 expression increased 20-fold (Fig. 8A and B).

Fig. 8. Cyclin Expression are Suppressed in Small-for-Size Liver Grafts: Reversal by AR.

Fig. 8

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Livers were harvested at 48 h after transplantation. Cyclin D1 (CyD1), cyclin E (CyE) and actin were detected by immunoblotting. Panel A shows representative gels (n = 4/group). Panel B plots mean CyD1/actin ratios determined by densitometry. Panel C plots CyE/actin. a, p<0.05 vs. sham-operation; b, p<0.05 vs FSG (100%); c, p<0.05 vs HSG (50%); d, p<0.05 vs TSG (30%) (n = 4/group).

Cyclin E (CyE), another cyclin that controls cell cycle progression, was expressed at low levels in sham-operated livers and FSG (Fig. 8A). CyE expression increased 5-fold in HSG but remained at low levels in TSG (Fig. 8A and C). AR treatment also increased CyE expression markedly in TSG (Fig. 8A and C). Overall, CyD1 and CyE expression paralleled closely with the proliferative responses of liver grafts under various conditions.

DISCUSSION

Suppressed AR Formation Contributes to Failure of Small-for-Size Liver Grafts

Insufficient liver volume is the major cause of small-for-size graft syndrome which decreases survival after PLT (5;31;32). Therefore, major transplant centers reject potential donors if estimated graft weight/recipient body weight is less than 0.8–1.0 or graft weight/recipient standard liver weight<30%-40% (32). Dual graft LT (two liver grafts from two donors into one recipient) was developed to increase the graft mass (33); however, it puts two donors at risk. A recent attempt was made to avoid small-for-size syndrome in living donor LT by increasing the donor’s liver mass before liver donation by eating a high-protein and high-carbohydrate diet for 3 months to increase calorie intake (34). Whether this approach increases the risk of steatosis and diabetes remains to be explored.

The mechanisms of small-for-size graft failure remain unclear. Alterations in hemodynamics, sheer stress, microcirculatory disturbances and increased reactive oxygen species (ROS) formation in small-for-size grafts may contribute to the grafts injury (11;35;36). Mitochondrial dysfunction occurs in small-for-size grafts, compromising energy supply (6;10). STY720, which internalizes and downregulates sphingosine-1-phosphate receptors, decreased injury after PLT (37).

The liver has a robust capacity to regenerate under most conditions which allows rapid recovery of liver mass and function after liver injury or loss of liver mass (e.g. liver resection). However, liver regeneration is suppressed in small-for-size grafts (6;9;38) which may contribute to graft dysfunction. Consistent with previous findings, we observed a markedly decreased liver regeneration in TSG which was associated poorer liver function and survival (Figs. 3–4).

Liver regeneration is tightly regulated by variety of transcription factors, cytokines and growth factors (13;15). In the early stage after PHX, a transcriptional shift occurs, which consists of activation of genes and transcriptional factors, and these massive changes in immediate early gene expression prime quiescent hepatocytes, making them responsive to growth factors, such as HGF and ligands of EGFR (13;15;39). Growth factors stimulate cell cycle progression by two major growth factor signaling systems: EGFR ligands/receptors/coreceptors and HGF/c-Met (12–15). Cytokines (e.g., TNFα and IL-6) activate transcription factors and also provide early signals triggering regeneration (12;40). Inhibition of transcription factor activation or decreased synthesis of pro-mitogenic cytokines and growth factors can decrease regeneration.

Previously we found that HGF, TNFα and IL-6 increased after rat PLT and peaked at about 5 h, an effect that was greater in QSG than in FSG and HSG (6). In this study, we confirmed that HGF, TNFα and IL-6 mRNAs increase to a greater extent in TSG (Fig. 2). Therefore, inhibited regeneration in small-for-size grafts was unlikely due to deficiency of pro-regenerative HGF, TNFα and IL-6 formation.

In this study, we further investigated the role of EGFR ligands AR, HB-EGF, EGF, and TGFα in suppressed regeneration of small-for-size liver grafts. After transplantation, AR increased markedly in HSG, which regenerated rapidly, but remained at low in TSG which failed to regenerate (Fig. 1, 3 and 4). AR neutralizing antibody blunted liver regeneration in HSG, whereas AR supplementation stimulated regeneration of TSG. Together, these findings are consistent with the conclusion that AR plays an important role in liver regeneration after PLT (Fig. 4).

Decreased AR was associated with lower AR mRNA in TSG, suggesting suppressed synthesis of AR in TSG (Fig. 1C). Signaling pathways that regulate AR formation include transcription factor WT1, protein kinases A and C, prostaglandin E2, Toll-like receptor-4, hypoxia-inducible factor-1α and ROS (41–43). However, we observed that Toll-like receptor-4 expression and ROS increased to a greater extent in TSG than in FSG (data not shown) (44). Therefore, Toll-like receptor-4 and ROS are unlikely the major factors contributing to suppressed AR formation. Future studies will be performed to investigate the mechanisms underlying suppressed AR expression in small-for-size grafts.

We also examined the possibility that AR was not released to act upon the EGFR. AR precursor is activated by a transmembrane metalloproteinase ADAM17 (20). ADAM17 was expressed at high levels in both HSG and TSG (Fig. 1). Therefore, decreased liver regeneration is not due to ADAM17 deficiency.

In this model, it does not appear that EGF and HB-EGF play a role in suppressed TSG regeneration since the levels of neither growth factor increased significantly following PLT. It is also unlikely that TGFα plays a limiting role in suppressed TSG regeneration because TGFα levels increased equally following HSG and TSG. Together, these data are consistent with previous studies which demonstrated that removal of AR decreased liver regeneration whereas deletion of some other EGFR ligands did not (14;19), and the findings support the hypothesis that decreased AR synthesis is responsible, at least in part, for the suppressed regeneration of small-for-size liver grafts.

There are other examples of ligands that do not appear to be interchangeable. For example, TGFα is a strong mitogen for hepatocytes in culture and increased modestly after PHX (45;46). However, TGFα knock-out mice have essentially normal liver regeneration and liver embryonic development (47). In HB-EGF-deficient mice, liver regeneration decreased moderately in the early stage but increased in late stage after PHX (48).

EGFR Mediates Stimulation of Small-for-Size Graft Regeneration by AR

Treatment with RNAi against the EGFR decreased cell proliferation after PHX in rats (49), indicating that EGFR plays an essential role in regeneration. In the present study, EGFR activation was increased in HSG but not in TSG (Fig. 6), consistent with the regenerative responses in HSG and the lack of one in TSG (Figs. 3–4). Inhibition of EGFR by PD153035 significantly decreased liver regeneration in HSG (Fig. 6), confirming the importance of EGFR in regeneration of partial liver grafts.

AR treatment increased EGFR phosphorylation in TSG to the levels of HSG, indicating that EGFR retains its responsiveness to AR stimulation (Fig. 6). Thus, AR could be used in small-for-size transplantation to stimulate liver regeneration. AR exclusively binds and activates the EGFR but not other members of the EGFR family of receptors like ErbB4 or ErbB3, although it can promote receptor heterodimerization (ErbB-2 with ErbB-1) (43;50). However, ErbB-2 is mainly expressed in tumors. EGFR inhibition largely blocked AR-stimulated liver regeneration in TSG (Fig. 6), suggesting that the promitotic effects of AR in small-for-size grafts are primarily mediated by EGFR. Whether other EGFR lignads have similar therapeutic effects for small-for-size grafts is beyond the scope of this study and will be investigated in the future.

Role of EGFR Downstream Signaling Pathways in Stimulation of Small-for-Size Graft Regeneration by AR

Ligand binding to EGFR activates a number of downstream signaling pathways (21;22). Activation of the PI3K pathway produces phosphatidylinositol (3,4,5)-P3 which activates different kinases such as phosphoinositide dependent kinase1 (PDK1) and protein kinase B. PDK1-deficiency suppressed regeneration after PHX (51). Akt, an important downstream effector of PI3K, activates mTOR and p70S6 kinase which in turn regulates the 40S ribosomal protein S6 to control protein synthesis and cell proliferation. Inhibition of mTOR decreased DNA synthesis after PHX (52). Deletion of S6 protein led to a profound deficit in DNA replication and alterations in cyclin E induction after PHX (53).

In this study, activation of PI3K, mTOR and its downstream effector p70S6K all increased in HSG, not in TSG, and was restored with AR treatment (Fig. 7). The PI3K/mTOR pathway is highly sensitive to nutrient/energy alterations (13;54). Small-for-size liver grafts face significantly higher metabolic challenges and need to meet the demands for significant nucleotide and protein synthesis for cell division. However, ATP production decreases substantially in small-for-size liver grafts (6). Therefore, compromised energy status may also contribute to decreased activity in PI3K/mTOR pathway.

EGFR also leads to activation of ERK and JNK pathways (13;55–58). Both ERK and JNK mediate AR-induced hepatocyte proliferation (19). Thus, interruption of JNK and ERK signaling might be involved in inhibited regeneration of small partial liver grafts. The ERK kinases are activated by EGFR through G proteins in the Ras/Raf/ERK pathway. Activated ERK kinases regulate a variety of transcription factors thus controlling transcription of important cell cycle genes (59). Blockade of ERK activation suppressed synthesis of CyE and A as well as CDK2 activation (60).

Inhibition of JNK decreases expression of CyD1, a molecule that drives hepatocytes to enter the cell cycle (61), and suppresses hepatocyte mitosis after PHX (28). JNK activation is controlled by TNFα; however, TNFα expression is increased in small-for-size grafts (6). Therefore, inhibition of JNK activation is not due to lack of TNFα. EGFR also regulates JNK activation. Indeed, JNK and ERK phosphorylation and expression of cyclins were inhibited in TSG (Figs. 7–8), which was associated with suppressed EGFR phosphorylation and liver regeneration. AR treatment recovered the activation of ERK and JNK as well (Figs. 7–8).

It appears that multiple, redundant pathways mediate the proliferative effects of AR/EGFR in partial liver grafts. In our previous studies we also observed increased formation of TGFβ, an inhibitor of liver regeneration in small-for-size liver grafts (7). Suppression of the TGFβ signaling or stimulation of EGFR signaling both improved regeneration of small-for-size liver grafts. These results indicate that liver regeneration is controlled by a delicate balance of pro-regenerative and regeneration-inhibitory factors.

Taken together, we observed decreased AR formation and subsequent inhibited EGFR signaling in small-for-size liver grafts. This decreased AR formation is associated with suppressed liver regeneration and failure of small-for-size liver grafts. However, small-for-size grafts retain their responsiveness to AR stimulation. Therefore, AR supplementation could be a promising therapy for small-for-size syndrome. Of note, the therapeutic effect of AR was observed in a model in which the hepatic artery was not reconstructed. The importance of reconstruction of hepatic artery has been controversial. One study showed that hepatic artery reconstruction improved survival and decreased transaminase release after mouse LT (62). By contrast, other studies showed that at 1 and 100 days after mouse LT, no differences in survival, injury and immunologic responses were observed with or without rearterization (63). Transplantation of HSG without rearterization achieves high survival, as confirmed in the current study (64) (Fig. 5). Moreover, regardless the absence of hepatic artery reconstruction, AR improved the outcome of small-for-size LT, indicating that its mitotic effect is independent of hepatic arterial blood supply.

Supplementary Material

Supp Table S1 & Figure S1-S3

Acknowledgments

This study was supported, in part, by Grants DK70844, DK70844S1, DK084632, and DK037034 from the National Institutes of Health.

LIST OF ABBREVIATIONS

ADAM17

ADAM metallopeptidase domain 17

Akt

protein kinase B

ALT

alanine aminotransferase

AP-1

activator protein-1

AR

amphiregulin

BrdU

5-bromo-2′-deoxyuridine

C/EBPβ

CCAAT-enhancer-binding protein-β

CDK

cyclin-dependent kinase

CyD1

cyclin D1

CyE

cyclin E

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

ERK

extracellular-signal-regulated kinases

FOXD3

forkhead box D3

FSG

full-size grafts

HB-EGF

heparin-binding epidermal growth factor

HGF

hepatic growth factor

hpf

high power field

HPRT

hypoxanthine phospho-ribosyl-transferase

HSG

half-size grafts

IL-6

interleukin-6

JNK

c-Jun-N-terminal kinase

LT

liver transplantation

mTOR

mammalian target of rapamycin

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

PCNA

proliferating cell nuclear antigen

PDK1

phosphoinositide dependent kinase1

PHX

partial hepatectomy

PI3K

phosphoinositide kinase-3

PLT

partial liver transplantation

p70S6K

p70S6 kinase

qPCR

quantitative real-time polymerase chain reaction

ROS

reactive oxygen species

STAT3

signal transducer and activator of transcription 3

TGFα

transforming growth factor-α

TGFβ

transforming growth factor-β

TNFα

tumor necrosis factor-α

TSG

thirty percent-size grafts

TUNEL

terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

Footnotes

DISCLOSURE

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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Supplementary Materials

Supp Table S1 & Figure S1-S3
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