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Compensatory recovery of liver mass by Akt-mediated hepatocellular hypertrophy in liver-specific STAT3-deficient mice
Journal of Hepatology 43 (2005) 799–807 www.elsevier.com/locate/jhep
Compensatory recovery of liver mass by Akt-mediated hepatocellular hypertrophy in liver-specific STAT3-deficient mice Sanae Haga1, Wataru Ogawa2, Hiroshi Inoue2, Keita Terui3, Tetsuya Ogino4, Rumi Igarashi1, Kiyoshi Takeda5, Shizuo Akira6, Shin Enosawa7, Hiroyuki Furukawa1, Satoru Todo1, Michitaka Ozaki1,7,* 2
1 Department of Surgery, Hokkaido University Graduate School of Medicine, Faculty of Medicine, Sapporo, Japan Division of Diabetes and Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan 3 Department of Pediatric Surgery, Chiba University Graduate School of Medicine, Chiba, Japan 4 Department of Pathology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan 5 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan 6 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 7 Bioengineering Laboratory, Department of Innovative Surgery, National Research Institute for Child Health and Development, Tokyo, Japan
Background/Aims: Liver regeneration following hepatectomy is complicated and involves a variety of interacting factors. The present study was designed to study the roles of proliferation and hypertrophy of hepatocytes in liver regeneration following hepatectomy in liver-specific STAT3-knockout (LS3-KO) mice lacking mitogenic activity. Methods: Partial hepatectomy was performed in LS3-KO and control mice. Liver regeneration was estimated by the liver weight, cell proliferation and cell size, and the related cellular signals were analyzed. Results: Proliferation of hepatocytes following PH was markedly suppressed in LS3-KO mice with reduced cyclinD1 transcript. However, liver mass recovered sufficiently following PH in LS3-KO mice almost equal to that of control mice. Analysis of hepatocellular growth revealed that cell size following hepatectomy was significantly larger in LS3KO mice than in control mice. Hepatectomy induced immediate but transient phosphorylation of Akt, p70S6K, mTOR and GSK-3b in LS3-KO mice much more than in control mice. Additionally, adenoviral transfection of dominant negative mutant of Akt to control and LS3-KO mice led to insufficient liver regeneration following hepatectomy. Conclusions: PI3-K/Akt-mediated responsive hepatocellular hypertrophy may be essential for liver regeneration following hepatectomy and sufficiently compensated liver regeneration even in STAT3-deficient liver, in which cell proliferation is impaired. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Hepatectomy; Cell size; Cell proliferation; Liver regeneration; mTOR; GSK-3; p70S6K 1. Introduction Liver regeneration is a physiological phenomenon of quantitative recovery from loss of liver mass to compensate for impaired hepatic function. Liver has a unique ability to restore lost volume, which rarely occurs in other organs
Received 8 November 2004; received in revised form 29 March 2005; accepted 29 March 2005; available online 31 May 2005 * Corresponding author. Tel.: C81 11 706 7063; fax: C81 11 706 7064. E-mail address: [email protected] (M. Ozaki).
[1–3]. Normal adult hepatocytes are usually quiescent with potential ability to replicate, which can be triggered easily by certain kinds of stimuli. After surgical procedures to reduce liver mass, such as partial hepatectomy (PH) or live donor-liver transplantation, rapid enlargement of residual or graft liver commonly takes place to restore liver function. Clinically, liver regeneration has important implications because many of therapeutic strategies depend on the ability of the liver to self-replicate. Poor or insufficient liver regeneration may be potentially fatal for these patients [4–6]. Therefore, understanding the bio-physiological
0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2005.03.027
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features of liver regeneration and developing the means to control this process could lead to clinical benefits, such as decreased morbidity and mortality due to poor regeneration, and a wider range of effective hepato-surgical treatment options for patients. The best-studied model of liver regeneration is 70% partial hepatectomy (PH) in rodents. It is well known that most of the remaining hepatocytes rapidly enter into the cell cycle; DNA synthesis initially takes place at 12–16 h post-hepatectomy and peaks at 24 h, and eventually restoration of the original liver mass occurs in 7–10 days [7–9]. A considerable amount of research has been performed to determine the mechanism of cell proliferation underlying liver regeneration following PH, particularly with respect to the initiation, maintenance and termination of cell proliferation; however cell growth has not yet been thoroughly investigated. Signal transducer and activator of transcription-3 (STAT3) is ubiquitously expressed and transiently activated by a variety of different ligands, including interleukin-6 (IL-6), leukemia-inhibitory factor (LIF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), as well as by a number of oncogenic receptor- and non-receptor-tyrosine kinases [10–14]. STAT3 was first described based on the DNA-binding activity of IL-6-stimulated hepatocytes, capable of selectively interacting with an enhancer element in the promoter of acute-phase genes, known as acute-phase response elements [15–17]. The physiological functions of STAT3 have been extensively studied, and it is known to play a crucial role in the development of various organs and in cell proliferation [14]. Though targeted disruption of the STAT3 gene actually leads to early embryonic lethality [18], a STAT3-conditional knockout mouse (liver, lymphocyte) [18,19] has been developed. In liver-specific STAT3-KO (LS3-KO) mice, no abnormalities of liver development or structure were observed, but they showed reduced mitogenic response following PH [19]. Many other studies have also reported that STAT3 influences several important immediate early responsive genes following PH [13,20], and it has recently been shown to target genes other than mitogenic genes [21,22]. The PI3-K/Akt pathway is well known as a ‘survival signaling pathway’. This pathway targets various molecules involved in anti-apoptosis, anti-oxidative defenses and protein synthesis [23–27]. Recent evidence has shown that the PI3-K/Akt pathway and its downstream signaling molecules are responsible for determining cell size [28–35]. Many of these studies utilized transgenic animals of the targeted genes to show the crucial roles of Akt and their downstream molecules, such as mTOR, p70S6K and GSK-3, in determining the specific cell size in each organ. However, the relevance of their roles in cell size in an acute response such as PH is still unclear. In the present study, we examined the involvement of the PI3-K/Akt pathway in the acute response of liver regeneration following PH in liver-specific STAT3-knockout mice. We focused on the potential role of Akt and downstream
signals in compensatory hepatocellular hypertrophy during liver regeneration where cell proliferation is impaired.
2. Materials and methods 2.1. Generation of liver-specific STAT3-knock out (LS3-KO) mice We generated LS3-KO mice by breeding STAT3-flox mice [36], with albumin-Cre transgenic (Alb-Cre) mice [37], which express Cre recombinase specifically in the liver under the control of albumin promoter. To increase the efficiency of STAT3 disruption, we also introduced a STAT3 null allele over the floxed allele by crossing with STAT3 heterozygous knockout (Stat3C/K) mice [18]. We crossed STAT3C/K mice with Alb-Cre mice and generated STAT3C/K; Alb-Cre mice and bred these mice with mice harboring homozygous STAT3 floxed allele (STAT3flox/flox); the resultant STAT3flox/K; Alb-Cre mice were used as LS3-KO mice. STAT3flox/K mice obtained from the same breeding were used as control mice.
2.2. Animal experiments C57BL/6 mice (male, 20–25 g) were used for simple 70% PH experiments. Anesthesia was induced with an intraperitoneal injection of Nembutal (pentobarbital sodium, 60 mg/100 g body weight). Mice were fasted overnight prior to the experiments. After laparotomy, the left and medianliverlobesweresurgicallyresected.Themiceweresacrificedand liver specimens were collected at several time points before and after hepatectomy, and the liver/body weight ratios were measured to assess the recovery of liver mass. The animals were maintained under standard conditions and treated according to the Guidelines for the Care and Use of Laboratory Animals of the National Research Institute for Child Health and Development.
2.3. Measurement of cell size The cell size of hepatocytes was measured from electron photomicrographs of liver tissue. Liver samples were trimmed into small pieces and fixed in 1% glutaraldehyde and 2% formaldehyde in 0.1 M PBS (pH 7.4) at room temperature overnight, and post-fixed in 2% osmium tetroxide for 2 h. The samples were dehydrated in ascending concentrations of ethanol and embedded in epoxy resin. Ultrathin sections (70–80 nm) were prepared and stained with uranyl acetate and lead citrate, and observed using a highresolution electron microscope (JEM-1200, JEOL, Tokyo, Japan). Individual hepatocytes were outlined and cross-sectional area was determined with a computer-assisted image analysis system (LSM Image Browser, Carl Zeiss GmBH, Jena, Germany). Cell areas of at least 100 hepatocytes were calculated in triplicate using different sections in each group.
2.4. Western blot analysis For Western blot analysis, 30 mg of whole liver protein extract was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The following antibodies were used as primary antibodies; STAT3/phospho-STAT3 (Santa Cruz, CA), Akt/phospho-Akt (Thr and Ser), p70S6K/phospho-p70S6K, mTOR/phospho-mTOR, GSK3/phosphoGSK3 (Cell Signaling, MA).
2.5. Akt activity assay Akt activity assay was performed using an Akt kinase assay kit (Cell Signaling) according to the manufacturer’s recommendation. Briefly, 1.5 mg of whole liver protein extract was immunoprecipitated with immobilized Akt antibody. Pellets were reacted with GSK-3a/b fusion protein in the presence of ATP, and were then electrophoresed, and blotted. Phosphorylated GSK-3a/b fusion protein was detected with anti- phosphoGSK-3a/b (Ser21/9) antibody (Cell Signaling, MA).
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2.6. Cell proliferation assay In order to assess proliferation of hepatocytes following PH, mitotic hepatocytes and PCNA-positive hepatocytes were counted. Liver tissues were removed prior to and 3 days after hepatectomy, fixed in 10% buffered formalin and paraffin-embedded. Hematoxylin and eosin (H and E) staining and immunohistochemical staining with anti-PCNA were performed. Mitotic or PCNA-positive hepatocytes were counted for 500 hepatocytes at least three times in different sections in each group.
2.7. RT-PCR for cyclinD1 and cycline Total RNA was extracted using Isogen reagent (Nippon Gene, Tokyo, Japan). First-strand cDNA synthesis was performed with reverse transcriptase, 5 mg of total RNA, and oligo (dT) primers. The cDNA was amplified by PCR with mouse cyclinD1, cyclinE and glyceraldehyde3-phosphate dehydrogenase (GAPDH). Primer pairs of cyclinD1, cyclinE and GAPDH were 5 0 -CGCGTACCCTGACACCAATCT-3 0 and 5 0 CCGCATGGATGGCACAATCTC-3 0 , 5 0 -AGAGAGACTCGACGGAC CAC-3 0 , 5 0 -TAGGCCACTTGGACATAGAC-3 0 and 5 0 -TCCTGCAC CACCAACTGCTTAG-3 0 and 5 0 -CAGATCCACAACGGATACATTGG3 0 , respectively. PCR products were separated on 1% agarose gels.
2.8. Adenoviral vectors (LacZ and Akt-AA) Adenovirus coding for b-galactosidase was used as a control vector. Akt-AA, where Thr308 and Ser473 of HA-tagged rat Akt1 were replaced by alanine, was used to inactivate Akt by inhibiting phosphorylation at Thr308 and Ser473 [38]. Adenoviruses coding dominantly negative mutant of Akt (AxCAAkt-AA) and b-galactosidase (AdLacZ) were injected intravenously at 2!109 pfu/body, 3 days prior to the experiments.
2.9. Apoptosis assay Cell Death Detection-ELISA Plus (Roche Diagnostics Corp., Basel, Switzerland) was used for the quantitative evaluation of apoptotic cells in liver tissue. Lysates of liver tissue were directly applied for this assay (40 mg of protein) and the assay procedure was performed according to the manufacture’s recommendations. Anti-Fas (Jo2, BD PharMingen, San Diego, CA, USA) was intraperitoneally injected to mice (0.3 mg/g body weight) for inducing apoptosis, and the liver specimen was used as a positive control.
2.10. Statistical analysis Results are expressed as meansGSEM. Statistical analyses were performed with Fishers’ test, and p values less than 0.05 were considered significant.
Fig. 1. Liver regeneration after 70% partial hepatectomy (PH) in C57BL/6 mice. (a) Changes of liver/body weight ratios after PH, (b) western blot analysis for total and phospho-STAT3 and-Akt after PH. At least three mice were sacrificed to measure liver/body weight ratios at each time point after PH.
the protein levels of either STAT3 or Akt in the liver tissue. Interestingly, both STAT3 and Akt, which were activated after PH, were inactivated until the liver mass began to recover (24–48 h after PH) (Fig. 1b). Analysis of liver specific STAT3-knockout (LS3-KO) mice. Liver weight did not differ significantly between LS3KO and control mice (Fig. 2a). Histological analysis of the liver of LS3-KO mice also revealed morphologically normal liver structure and cells (Fig. 2b), and STAT3 protein was deficient specifically in the liver in LS3-KO mice (Fig. 2c). 3.2. Recovery of liver mass after PH in LS3-KO mice
3. Results 3.1. Liver regeneration and phosphorylation of STAT3 and Akt after 70% partial hepatectomy (PH) Measurement of the residual liver/body weight following PH revealed that liver mass began to recover 24–48 h after PH and continued for at least 2 weeks (Fig. 1a). Activation of STAT3, assessed by tyrosine-phosphorylation of STAT3, was observed immediately after PH and recovered to pre-operative level within 24 h. Akt, assessed by threonine-/serine-phosphorylation of Akt and total Akt, was also weakly activated even in the quiescent state, and was activated immediately after PH. PH did not affect
The ratios of liver/body weight following PH are shown in Fig. 3. Interestingly, no significant difference was observed in liver mass recovery between control and LS3-KO mice up to at least two weeks after PH. 3.3. Impaired hepatocyte proliferation after partial hepatectomy in LS3-KO mice Histological examination of the remnant liver revealed the presence of numerous mitotic hepatocytes in the regenerating liver of control mice 72 h after PH, but very few were observed in the LS3-KO mice (Fig. 4a). Interestingly, some mitotic hepatocytes were observed 14 days after PH only in the liver of LS3-KO mice,
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the liver of control mice, but not in LS3-KO mice; however, it was not detected in the quiescent state in either group of mice (Fig. 4b). CyclinE was constitutively expressed in the liver of both groups of mice with or without PH. 3.4. Cell size of hepatocytes after PH in LS3-KO mice In order to understand the mechanism of normal recovery of liver mass following PH in LS3-KO mice, which lack the ability for hepatocyte proliferation, we next measured the cell size of hepatocytes. Electron photomicrograph of hepatocytes 72 h after PH revealed that hepatocytes were larger in LS3-KO mice than in control mice (Fig. 5a). Without PH, the size of hepatocytes did not differ between control and LS3-KO mice (Fig. 5b). Three days after PH, the cell size of hepatocytes in both groups increased significantly; however, the increase was significantly greater in the LS3-KO mice more than in the control mice. The increased cell size induced by PH recovered within normal range until 14 days after PH in both mice. 3.5. Activation of Akt, p70S6K, mTOR and GSK-3b after PH in LS3-KO mice Fig. 2. Analysis of liver specific STAT3-knockout (LS3-KO) mice. (a) Liver/body weight ratios of control and LS3-KO mice (male, 20–25 g body weight), (b) liver histology of control and LS3-KO mice (H and E, original magnification!250), (c) western blot analysis of STAT3 protein in various organs of control and LS3-KO mice. Three mice were examined at each group, and the data in (b) and (c) are their representative examples. [This figure appears in colour on the web.]
indicating the marked delay of mitotic response of hepatocytes. Immunohistochemical staining with PCNA also confirmed the delayed mitotic activity of hepatocytes in LS3-KO mice. Many hepatocytes showed mild or moderate PCNA-positivity 14 days post-PH in LS3-KO mice, though PCNA-positive cells almost disappeared in the control liver. (Fig. 4a). CyclinD1 transcript was detected 4 h after PH in
Fig. 3. Liver regeneration after PH in LS3-KO mice. Recovery of liver mass was assessed by liver/body weight ratios (%) up to 14 days after hepatectomy. Three mice were examined at each group.
We next examined the activation of Akt and downstream molecules in order to understand their roles in the regulation of cell size (Fig. 6). Akt, p70S6K, mTOR and GSK-3b were weakly phosphorylated before PH. By 4 h after PH, these molecules were phosphorylated to a greater extent in LS3-KO mice than in control mice. The activation of these molecules recovered to basal levels 3 days after PH, and was unchanged until 14 days after PH. 3.6. Impaired regeneration after PH in Akt-inactivated liver In order to confirm the role of Akt and down-stream molecules in liver regeneration in LS3-KO mice, we inhibited Akt activity by adenoviral gene transfer of dominant negative mutant of Akt (Akt-AA) to the liver. Intravenous injection of AxCAAkt-AA with 2!109 pfu/ body resulted in the expression of hemagglutinin (HA)tagged mutant Akt protein in liver (Fig. 7a). The Akt activity assay also revealed that Akt-AA dramatically reduced the Akt activity of liver tissue. Though Akt-AA did not increase the mortality of post-hepatectomized mice, Akt-AA effectively suppressed liver regeneration following PH in control as well as in LS3-KO mice (Fig. 7b). As expected, responsive hepatocellular hypertrophy was suppressed in livers transduced with Akt-AA in both control and LS3-KO mice (Fig. 7c). These data may indicate that hepatocellular hypertrophy is generally more crucial for the initial recovery of liver mass than cell proliferation following PH.
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Fig. 4. Hepatocyte proliferation 3 and 14 days after PH in LS3-KO mice. (a) Upper panel; liver sections of both control and LS3-KO mice were stained with Hematoxylin and Eosin (H and E), and immunohistochemically with PCNA (original magnification, !250). Arrowheads indicate mitotic hepatocytes. Lower panel; mitotic and PCNA-positive hepatocytes following PH were counted in at least 500 cells in triplicate using different sections. (b) RT-PCR analysis of mRNAs of cyclinD1 and cyclinE in liver after PH. [This figure appears in colour on the web.]
3.7. Role of Akt anti-apoptotic ativity in liver regeneration
4. Discussion
In order to examine whether the anti-apoptotic activity of Akt is involved in liver regeneration, we next examined apoptotic cells in liver tissue following PH. Apoptotic cells were not increased in liver tissue following PH in either control or LS3-KO mice (Fig. 8). In this PH model, at least, it does not seem that apoptosis plays an important role in liver regeneration, and hence Akt may not contribute to liver regeneration following PH in LS3-KO mice by preventing apoptosis.
Recent evidence indicates an essential role of certain molecules in determining cell size in genetically manipulated animals. It is now known that Akt, mTOR, p70S6K, and GSK-3b are all good candidates as determinants of the original cell size in various kinds of cells and/or organs [28–35]. However, the roles of these molecules in acute hepatic response to injury or volume loss are still unknown. Responsive proliferation of hepatocytes is considered to be the most important factor in the regeneration processes
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Fig. 5. Cell size of hepatocytes after PH in LS3-KO mice. Cell size of hepatocytes was measured using electron photomicrographs of hepatocytes before and 72 h/14d after PH. (a) Electron photomicrographs of liver tissue 72 h after PH in control and LS3-KO mice. (b) Quantitative analysis of cross-sectional area of hepatocytes before and 72 h after PH. Areas containing at least 100 hepatocytes were measured in triplicate in each group. Three mice were examined at each group.
following hepatectomy. Most studies have examined hepatocyte proliferation as the major mechanism for liver regeneration, and have focused mainly on the cell cycleregulatory system. In the present study, we focused on the role of cell growth using LS3-KO mice, which lack responsive cell proliferation in the post-hepatectomy state. Surprisingly, liver mass recovered very rapidly and
Fig. 7. Impaired liver regeneration after PH in LS3-KO mice with AktAA infection. (a) Adenovirus-mediated introduction of dominant negative mutant of Akt (Akt-AA) in liver was confirmed by detecting haemagglutinin (HA) tagged to Akt-AA (upper panel). Akt activity assay was performed in livers infected with LacZ and Akt-AA before and after PH (lower panel). (b) Changes of liver mass before and after PH in control and LS3-KO mice with Akt-DN. (c) Quantitative analysis of cross-sectional area of hepatocytes before and 72 h after PH. Areas containing at least 100 hepatocytes were measured in triplicate in each group.
Fig. 6. Activation (phosphorylation) of Akt, p70S6K, mTOR, and GSK3b after PH in control and LS3-KO mice. Western blot analysis of liver tissue was performed before, 4/72 h, and 14 d after PH.
normally even in LS3-KO mice as a result of the compensatory hypertrophy of hepatocytes. Akt and its downstream signals, such as mTOR, p70S6K, and GSK-3b, were all activated to a greater extent in LS3-KO mice than control mice. On the contrary, inhibition of Akt by dominant negative mutant of Akt resulted in suppression of liver regeneration both in control and LS3-KO mice. Together, these results suggest that Akt and downstream signals play
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Fig. 9. Serum levels of albumin in control and LS3-KO mice after PH. Fig. 8. Apoptosis after PH in control and LS3-KO mice. Enzyme-linked immunosorbent assay (ELISA) was performed to detect apoptosis before and after PH. Anti-Fas (Jo2) was intraperitoneally injected to mice (0.3 mg/g body weight) for inducing apoptosis, and was used as a positive control. a: P!0.05 vs. control and LS3-KO.
a critical role in liver regeneration by regulating cell size. This idea may be supported by the observation that constitutively active mutant of Akt (myristoilated Akt, myr-Akt) caused enlargement of liver mass without inducing cell proliferation when it was adenovirally overexpressed in liver [39]. We also observed the same effect of myr-Akt upon liver mass. Replication-deficient adenovirus vector coding myristoilated Akt gene (Myr-Akt) and LacZ were intravenously infected at 1!108 pfu per body. Myr-Akt enlarged liver by 30% without inducing cell proliferation, compared with LacZ-infected liver 3 days after adenoviral infection. PDK1 and putative PDK2 specifically phosphorylates Akt at Thr308 and Ser473 respectively, and phosphorylation of both sites contributes to the full activation of Akt [40]. In Fig. 1, Akt is somehow phosphorylated at Ser473 even in untreated mice, though both sites of Akt were phosphorylated markedly and immediately in response to PH. This may mean that these two kinases, regulated independently, phosphorylate Akt and contribute to its activation in liver. Though Akt and downstream signals seem to be definitely involved in regulating cell size in liver, the underlying mechanism is still unclear. One possible mechanism may involve the important role of activated Akt (and mTOR) in producing cellular proteins, which leads to hepatocellular hypertrophy [30,41]. In support of this possibility, the serum level of albumin remained within the normal range following PH in LS3-KO mice, whereas it was greatly reduced in control mice (Fig. 9). Activated Akt may have contributed to maintaining high serum albumin levels after PH. The increased protein production by hepatocytes in response to activated Akt might contribute to the responsive increase in cell size. Very interestingly, some mitotic hepatocytes were observed in livers of LS3-KO mice 14 days after PH, though they were not observed in early stage after PH. On the contrary, no mitosis was found in control liver 14 days after PH. Knockout of STAT3 in liver may have led to the extremely delayed response of hepatic mitogenesis after PH,
and Akt-mediated hepatocyte hypertrophy transiently compensated liver mass. Increased cell size in liver of LS3-KO mice 3 days after PH recovered within normal range until 14 days after PH. So it indicates that signalmediated hepatocellular hepertrophy occurred and maintained liver mass until mitosis of hepatocytes starts. Because apoptotic cell death may occur following PH and cause impaired liver regeneration, particularly under certain pathological conditions or as a result of excessive hepatic resection [42–46], and since the anti-apoptotic property of Akt has been widely documented [24–26,47], we investigated the possible involvement of Akt-mediated anti-apoptotic activity in the recovery of liver mass. We evaluated hepatic apoptosis in regenerating liver tissue and found that apoptotic cell death did not increase in control or LS3-KO mice following PH. This finding indicates that Akt contributes to liver regeneration not through its antiapoptotic property, but through other mechanisms. Apoptosis of hepatocytes does not appear to play a central role in maintaining liver regeneration. In conclusion, Akt and downstream signals may play crucial roles in liver regeneration by increasing cell size. Liver regeneration (recovery of liver mass) occurred normally without cell proliferation in LS3-KO mice, but was significantly impaired by inactivation of Akt. Responsive hepatocellular hypertrophy may be indispensable for the recovery of liver mass following hepatectomy under both normal and pathological conditions. Further studies are required to clarify the role of cell proliferation and growth during liver regeneration, and to determine their regulatory mechanisms. The present study, however, may indicate that responsive hepatocellular hypertrophy is more important than cell proliferation for acute liver regeneration.
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Compensatory recovery of liver mass by Akt-mediated hepatocellular hypertrophy in liver-specific STAT3-deficient mice Sanae Haga1, Wataru Ogawa2, Hiroshi Inoue2, Keita Terui3, Tetsuya Ogino4, Rumi Igarashi1, Kiyoshi Takeda5, Shizuo Akira6, Shin Enosawa7, Hiroyuki Furukawa1, Satoru Todo1, Michitaka Ozaki1,7,* 2
1 Department of Surgery, Hokkaido University Graduate School of Medicine, Faculty of Medicine, Sapporo, Japan Division of Diabetes and Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan 3 Department of Pediatric Surgery, Chiba University Graduate School of Medicine, Chiba, Japan 4 Department of Pathology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan 5 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan 6 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 7 Bioengineering Laboratory, Department of Innovative Surgery, National Research Institute for Child Health and Development, Tokyo, Japan
Background/Aims: Liver regeneration following hepatectomy is complicated and involves a variety of interacting factors. The present study was designed to study the roles of proliferation and hypertrophy of hepatocytes in liver regeneration following hepatectomy in liver-specific STAT3-knockout (LS3-KO) mice lacking mitogenic activity. Methods: Partial hepatectomy was performed in LS3-KO and control mice. Liver regeneration was estimated by the liver weight, cell proliferation and cell size, and the related cellular signals were analyzed. Results: Proliferation of hepatocytes following PH was markedly suppressed in LS3-KO mice with reduced cyclinD1 transcript. However, liver mass recovered sufficiently following PH in LS3-KO mice almost equal to that of control mice. Analysis of hepatocellular growth revealed that cell size following hepatectomy was significantly larger in LS3KO mice than in control mice. Hepatectomy induced immediate but transient phosphorylation of Akt, p70S6K, mTOR and GSK-3b in LS3-KO mice much more than in control mice. Additionally, adenoviral transfection of dominant negative mutant of Akt to control and LS3-KO mice led to insufficient liver regeneration following hepatectomy. Conclusions: PI3-K/Akt-mediated responsive hepatocellular hypertrophy may be essential for liver regeneration following hepatectomy and sufficiently compensated liver regeneration even in STAT3-deficient liver, in which cell proliferation is impaired. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Hepatectomy; Cell size; Cell proliferation; Liver regeneration; mTOR; GSK-3; p70S6K 1. Introduction Liver regeneration is a physiological phenomenon of quantitative recovery from loss of liver mass to compensate for impaired hepatic function. Liver has a unique ability to restore lost volume, which rarely occurs in other organs
Received 8 November 2004; received in revised form 29 March 2005; accepted 29 March 2005; available online 31 May 2005 * Corresponding author. Tel.: C81 11 706 7063; fax: C81 11 706 7064. E-mail address: [email protected] (M. Ozaki).
[1–3]. Normal adult hepatocytes are usually quiescent with potential ability to replicate, which can be triggered easily by certain kinds of stimuli. After surgical procedures to reduce liver mass, such as partial hepatectomy (PH) or live donor-liver transplantation, rapid enlargement of residual or graft liver commonly takes place to restore liver function. Clinically, liver regeneration has important implications because many of therapeutic strategies depend on the ability of the liver to self-replicate. Poor or insufficient liver regeneration may be potentially fatal for these patients [4–6]. Therefore, understanding the bio-physiological
0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2005.03.027
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features of liver regeneration and developing the means to control this process could lead to clinical benefits, such as decreased morbidity and mortality due to poor regeneration, and a wider range of effective hepato-surgical treatment options for patients. The best-studied model of liver regeneration is 70% partial hepatectomy (PH) in rodents. It is well known that most of the remaining hepatocytes rapidly enter into the cell cycle; DNA synthesis initially takes place at 12–16 h post-hepatectomy and peaks at 24 h, and eventually restoration of the original liver mass occurs in 7–10 days [7–9]. A considerable amount of research has been performed to determine the mechanism of cell proliferation underlying liver regeneration following PH, particularly with respect to the initiation, maintenance and termination of cell proliferation; however cell growth has not yet been thoroughly investigated. Signal transducer and activator of transcription-3 (STAT3) is ubiquitously expressed and transiently activated by a variety of different ligands, including interleukin-6 (IL-6), leukemia-inhibitory factor (LIF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), as well as by a number of oncogenic receptor- and non-receptor-tyrosine kinases [10–14]. STAT3 was first described based on the DNA-binding activity of IL-6-stimulated hepatocytes, capable of selectively interacting with an enhancer element in the promoter of acute-phase genes, known as acute-phase response elements [15–17]. The physiological functions of STAT3 have been extensively studied, and it is known to play a crucial role in the development of various organs and in cell proliferation [14]. Though targeted disruption of the STAT3 gene actually leads to early embryonic lethality [18], a STAT3-conditional knockout mouse (liver, lymphocyte) [18,19] has been developed. In liver-specific STAT3-KO (LS3-KO) mice, no abnormalities of liver development or structure were observed, but they showed reduced mitogenic response following PH [19]. Many other studies have also reported that STAT3 influences several important immediate early responsive genes following PH [13,20], and it has recently been shown to target genes other than mitogenic genes [21,22]. The PI3-K/Akt pathway is well known as a ‘survival signaling pathway’. This pathway targets various molecules involved in anti-apoptosis, anti-oxidative defenses and protein synthesis [23–27]. Recent evidence has shown that the PI3-K/Akt pathway and its downstream signaling molecules are responsible for determining cell size [28–35]. Many of these studies utilized transgenic animals of the targeted genes to show the crucial roles of Akt and their downstream molecules, such as mTOR, p70S6K and GSK-3, in determining the specific cell size in each organ. However, the relevance of their roles in cell size in an acute response such as PH is still unclear. In the present study, we examined the involvement of the PI3-K/Akt pathway in the acute response of liver regeneration following PH in liver-specific STAT3-knockout mice. We focused on the potential role of Akt and downstream
signals in compensatory hepatocellular hypertrophy during liver regeneration where cell proliferation is impaired.
2. Materials and methods 2.1. Generation of liver-specific STAT3-knock out (LS3-KO) mice We generated LS3-KO mice by breeding STAT3-flox mice [36], with albumin-Cre transgenic (Alb-Cre) mice [37], which express Cre recombinase specifically in the liver under the control of albumin promoter. To increase the efficiency of STAT3 disruption, we also introduced a STAT3 null allele over the floxed allele by crossing with STAT3 heterozygous knockout (Stat3C/K) mice [18]. We crossed STAT3C/K mice with Alb-Cre mice and generated STAT3C/K; Alb-Cre mice and bred these mice with mice harboring homozygous STAT3 floxed allele (STAT3flox/flox); the resultant STAT3flox/K; Alb-Cre mice were used as LS3-KO mice. STAT3flox/K mice obtained from the same breeding were used as control mice.
2.2. Animal experiments C57BL/6 mice (male, 20–25 g) were used for simple 70% PH experiments. Anesthesia was induced with an intraperitoneal injection of Nembutal (pentobarbital sodium, 60 mg/100 g body weight). Mice were fasted overnight prior to the experiments. After laparotomy, the left and medianliverlobesweresurgicallyresected.Themiceweresacrificedand liver specimens were collected at several time points before and after hepatectomy, and the liver/body weight ratios were measured to assess the recovery of liver mass. The animals were maintained under standard conditions and treated according to the Guidelines for the Care and Use of Laboratory Animals of the National Research Institute for Child Health and Development.
2.3. Measurement of cell size The cell size of hepatocytes was measured from electron photomicrographs of liver tissue. Liver samples were trimmed into small pieces and fixed in 1% glutaraldehyde and 2% formaldehyde in 0.1 M PBS (pH 7.4) at room temperature overnight, and post-fixed in 2% osmium tetroxide for 2 h. The samples were dehydrated in ascending concentrations of ethanol and embedded in epoxy resin. Ultrathin sections (70–80 nm) were prepared and stained with uranyl acetate and lead citrate, and observed using a highresolution electron microscope (JEM-1200, JEOL, Tokyo, Japan). Individual hepatocytes were outlined and cross-sectional area was determined with a computer-assisted image analysis system (LSM Image Browser, Carl Zeiss GmBH, Jena, Germany). Cell areas of at least 100 hepatocytes were calculated in triplicate using different sections in each group.
2.4. Western blot analysis For Western blot analysis, 30 mg of whole liver protein extract was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The following antibodies were used as primary antibodies; STAT3/phospho-STAT3 (Santa Cruz, CA), Akt/phospho-Akt (Thr and Ser), p70S6K/phospho-p70S6K, mTOR/phospho-mTOR, GSK3/phosphoGSK3 (Cell Signaling, MA).
2.5. Akt activity assay Akt activity assay was performed using an Akt kinase assay kit (Cell Signaling) according to the manufacturer’s recommendation. Briefly, 1.5 mg of whole liver protein extract was immunoprecipitated with immobilized Akt antibody. Pellets were reacted with GSK-3a/b fusion protein in the presence of ATP, and were then electrophoresed, and blotted. Phosphorylated GSK-3a/b fusion protein was detected with anti- phosphoGSK-3a/b (Ser21/9) antibody (Cell Signaling, MA).
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2.6. Cell proliferation assay In order to assess proliferation of hepatocytes following PH, mitotic hepatocytes and PCNA-positive hepatocytes were counted. Liver tissues were removed prior to and 3 days after hepatectomy, fixed in 10% buffered formalin and paraffin-embedded. Hematoxylin and eosin (H and E) staining and immunohistochemical staining with anti-PCNA were performed. Mitotic or PCNA-positive hepatocytes were counted for 500 hepatocytes at least three times in different sections in each group.
2.7. RT-PCR for cyclinD1 and cycline Total RNA was extracted using Isogen reagent (Nippon Gene, Tokyo, Japan). First-strand cDNA synthesis was performed with reverse transcriptase, 5 mg of total RNA, and oligo (dT) primers. The cDNA was amplified by PCR with mouse cyclinD1, cyclinE and glyceraldehyde3-phosphate dehydrogenase (GAPDH). Primer pairs of cyclinD1, cyclinE and GAPDH were 5 0 -CGCGTACCCTGACACCAATCT-3 0 and 5 0 CCGCATGGATGGCACAATCTC-3 0 , 5 0 -AGAGAGACTCGACGGAC CAC-3 0 , 5 0 -TAGGCCACTTGGACATAGAC-3 0 and 5 0 -TCCTGCAC CACCAACTGCTTAG-3 0 and 5 0 -CAGATCCACAACGGATACATTGG3 0 , respectively. PCR products were separated on 1% agarose gels.
2.8. Adenoviral vectors (LacZ and Akt-AA) Adenovirus coding for b-galactosidase was used as a control vector. Akt-AA, where Thr308 and Ser473 of HA-tagged rat Akt1 were replaced by alanine, was used to inactivate Akt by inhibiting phosphorylation at Thr308 and Ser473 [38]. Adenoviruses coding dominantly negative mutant of Akt (AxCAAkt-AA) and b-galactosidase (AdLacZ) were injected intravenously at 2!109 pfu/body, 3 days prior to the experiments.
2.9. Apoptosis assay Cell Death Detection-ELISA Plus (Roche Diagnostics Corp., Basel, Switzerland) was used for the quantitative evaluation of apoptotic cells in liver tissue. Lysates of liver tissue were directly applied for this assay (40 mg of protein) and the assay procedure was performed according to the manufacture’s recommendations. Anti-Fas (Jo2, BD PharMingen, San Diego, CA, USA) was intraperitoneally injected to mice (0.3 mg/g body weight) for inducing apoptosis, and the liver specimen was used as a positive control.
2.10. Statistical analysis Results are expressed as meansGSEM. Statistical analyses were performed with Fishers’ test, and p values less than 0.05 were considered significant.
Fig. 1. Liver regeneration after 70% partial hepatectomy (PH) in C57BL/6 mice. (a) Changes of liver/body weight ratios after PH, (b) western blot analysis for total and phospho-STAT3 and-Akt after PH. At least three mice were sacrificed to measure liver/body weight ratios at each time point after PH.
the protein levels of either STAT3 or Akt in the liver tissue. Interestingly, both STAT3 and Akt, which were activated after PH, were inactivated until the liver mass began to recover (24–48 h after PH) (Fig. 1b). Analysis of liver specific STAT3-knockout (LS3-KO) mice. Liver weight did not differ significantly between LS3KO and control mice (Fig. 2a). Histological analysis of the liver of LS3-KO mice also revealed morphologically normal liver structure and cells (Fig. 2b), and STAT3 protein was deficient specifically in the liver in LS3-KO mice (Fig. 2c). 3.2. Recovery of liver mass after PH in LS3-KO mice
3. Results 3.1. Liver regeneration and phosphorylation of STAT3 and Akt after 70% partial hepatectomy (PH) Measurement of the residual liver/body weight following PH revealed that liver mass began to recover 24–48 h after PH and continued for at least 2 weeks (Fig. 1a). Activation of STAT3, assessed by tyrosine-phosphorylation of STAT3, was observed immediately after PH and recovered to pre-operative level within 24 h. Akt, assessed by threonine-/serine-phosphorylation of Akt and total Akt, was also weakly activated even in the quiescent state, and was activated immediately after PH. PH did not affect
The ratios of liver/body weight following PH are shown in Fig. 3. Interestingly, no significant difference was observed in liver mass recovery between control and LS3-KO mice up to at least two weeks after PH. 3.3. Impaired hepatocyte proliferation after partial hepatectomy in LS3-KO mice Histological examination of the remnant liver revealed the presence of numerous mitotic hepatocytes in the regenerating liver of control mice 72 h after PH, but very few were observed in the LS3-KO mice (Fig. 4a). Interestingly, some mitotic hepatocytes were observed 14 days after PH only in the liver of LS3-KO mice,
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the liver of control mice, but not in LS3-KO mice; however, it was not detected in the quiescent state in either group of mice (Fig. 4b). CyclinE was constitutively expressed in the liver of both groups of mice with or without PH. 3.4. Cell size of hepatocytes after PH in LS3-KO mice In order to understand the mechanism of normal recovery of liver mass following PH in LS3-KO mice, which lack the ability for hepatocyte proliferation, we next measured the cell size of hepatocytes. Electron photomicrograph of hepatocytes 72 h after PH revealed that hepatocytes were larger in LS3-KO mice than in control mice (Fig. 5a). Without PH, the size of hepatocytes did not differ between control and LS3-KO mice (Fig. 5b). Three days after PH, the cell size of hepatocytes in both groups increased significantly; however, the increase was significantly greater in the LS3-KO mice more than in the control mice. The increased cell size induced by PH recovered within normal range until 14 days after PH in both mice. 3.5. Activation of Akt, p70S6K, mTOR and GSK-3b after PH in LS3-KO mice Fig. 2. Analysis of liver specific STAT3-knockout (LS3-KO) mice. (a) Liver/body weight ratios of control and LS3-KO mice (male, 20–25 g body weight), (b) liver histology of control and LS3-KO mice (H and E, original magnification!250), (c) western blot analysis of STAT3 protein in various organs of control and LS3-KO mice. Three mice were examined at each group, and the data in (b) and (c) are their representative examples. [This figure appears in colour on the web.]
indicating the marked delay of mitotic response of hepatocytes. Immunohistochemical staining with PCNA also confirmed the delayed mitotic activity of hepatocytes in LS3-KO mice. Many hepatocytes showed mild or moderate PCNA-positivity 14 days post-PH in LS3-KO mice, though PCNA-positive cells almost disappeared in the control liver. (Fig. 4a). CyclinD1 transcript was detected 4 h after PH in
Fig. 3. Liver regeneration after PH in LS3-KO mice. Recovery of liver mass was assessed by liver/body weight ratios (%) up to 14 days after hepatectomy. Three mice were examined at each group.
We next examined the activation of Akt and downstream molecules in order to understand their roles in the regulation of cell size (Fig. 6). Akt, p70S6K, mTOR and GSK-3b were weakly phosphorylated before PH. By 4 h after PH, these molecules were phosphorylated to a greater extent in LS3-KO mice than in control mice. The activation of these molecules recovered to basal levels 3 days after PH, and was unchanged until 14 days after PH. 3.6. Impaired regeneration after PH in Akt-inactivated liver In order to confirm the role of Akt and down-stream molecules in liver regeneration in LS3-KO mice, we inhibited Akt activity by adenoviral gene transfer of dominant negative mutant of Akt (Akt-AA) to the liver. Intravenous injection of AxCAAkt-AA with 2!109 pfu/ body resulted in the expression of hemagglutinin (HA)tagged mutant Akt protein in liver (Fig. 7a). The Akt activity assay also revealed that Akt-AA dramatically reduced the Akt activity of liver tissue. Though Akt-AA did not increase the mortality of post-hepatectomized mice, Akt-AA effectively suppressed liver regeneration following PH in control as well as in LS3-KO mice (Fig. 7b). As expected, responsive hepatocellular hypertrophy was suppressed in livers transduced with Akt-AA in both control and LS3-KO mice (Fig. 7c). These data may indicate that hepatocellular hypertrophy is generally more crucial for the initial recovery of liver mass than cell proliferation following PH.
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Fig. 4. Hepatocyte proliferation 3 and 14 days after PH in LS3-KO mice. (a) Upper panel; liver sections of both control and LS3-KO mice were stained with Hematoxylin and Eosin (H and E), and immunohistochemically with PCNA (original magnification, !250). Arrowheads indicate mitotic hepatocytes. Lower panel; mitotic and PCNA-positive hepatocytes following PH were counted in at least 500 cells in triplicate using different sections. (b) RT-PCR analysis of mRNAs of cyclinD1 and cyclinE in liver after PH. [This figure appears in colour on the web.]
3.7. Role of Akt anti-apoptotic ativity in liver regeneration
4. Discussion
In order to examine whether the anti-apoptotic activity of Akt is involved in liver regeneration, we next examined apoptotic cells in liver tissue following PH. Apoptotic cells were not increased in liver tissue following PH in either control or LS3-KO mice (Fig. 8). In this PH model, at least, it does not seem that apoptosis plays an important role in liver regeneration, and hence Akt may not contribute to liver regeneration following PH in LS3-KO mice by preventing apoptosis.
Recent evidence indicates an essential role of certain molecules in determining cell size in genetically manipulated animals. It is now known that Akt, mTOR, p70S6K, and GSK-3b are all good candidates as determinants of the original cell size in various kinds of cells and/or organs [28–35]. However, the roles of these molecules in acute hepatic response to injury or volume loss are still unknown. Responsive proliferation of hepatocytes is considered to be the most important factor in the regeneration processes
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Fig. 5. Cell size of hepatocytes after PH in LS3-KO mice. Cell size of hepatocytes was measured using electron photomicrographs of hepatocytes before and 72 h/14d after PH. (a) Electron photomicrographs of liver tissue 72 h after PH in control and LS3-KO mice. (b) Quantitative analysis of cross-sectional area of hepatocytes before and 72 h after PH. Areas containing at least 100 hepatocytes were measured in triplicate in each group. Three mice were examined at each group.
following hepatectomy. Most studies have examined hepatocyte proliferation as the major mechanism for liver regeneration, and have focused mainly on the cell cycleregulatory system. In the present study, we focused on the role of cell growth using LS3-KO mice, which lack responsive cell proliferation in the post-hepatectomy state. Surprisingly, liver mass recovered very rapidly and
Fig. 7. Impaired liver regeneration after PH in LS3-KO mice with AktAA infection. (a) Adenovirus-mediated introduction of dominant negative mutant of Akt (Akt-AA) in liver was confirmed by detecting haemagglutinin (HA) tagged to Akt-AA (upper panel). Akt activity assay was performed in livers infected with LacZ and Akt-AA before and after PH (lower panel). (b) Changes of liver mass before and after PH in control and LS3-KO mice with Akt-DN. (c) Quantitative analysis of cross-sectional area of hepatocytes before and 72 h after PH. Areas containing at least 100 hepatocytes were measured in triplicate in each group.
Fig. 6. Activation (phosphorylation) of Akt, p70S6K, mTOR, and GSK3b after PH in control and LS3-KO mice. Western blot analysis of liver tissue was performed before, 4/72 h, and 14 d after PH.
normally even in LS3-KO mice as a result of the compensatory hypertrophy of hepatocytes. Akt and its downstream signals, such as mTOR, p70S6K, and GSK-3b, were all activated to a greater extent in LS3-KO mice than control mice. On the contrary, inhibition of Akt by dominant negative mutant of Akt resulted in suppression of liver regeneration both in control and LS3-KO mice. Together, these results suggest that Akt and downstream signals play
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Fig. 9. Serum levels of albumin in control and LS3-KO mice after PH. Fig. 8. Apoptosis after PH in control and LS3-KO mice. Enzyme-linked immunosorbent assay (ELISA) was performed to detect apoptosis before and after PH. Anti-Fas (Jo2) was intraperitoneally injected to mice (0.3 mg/g body weight) for inducing apoptosis, and was used as a positive control. a: P!0.05 vs. control and LS3-KO.
a critical role in liver regeneration by regulating cell size. This idea may be supported by the observation that constitutively active mutant of Akt (myristoilated Akt, myr-Akt) caused enlargement of liver mass without inducing cell proliferation when it was adenovirally overexpressed in liver [39]. We also observed the same effect of myr-Akt upon liver mass. Replication-deficient adenovirus vector coding myristoilated Akt gene (Myr-Akt) and LacZ were intravenously infected at 1!108 pfu per body. Myr-Akt enlarged liver by 30% without inducing cell proliferation, compared with LacZ-infected liver 3 days after adenoviral infection. PDK1 and putative PDK2 specifically phosphorylates Akt at Thr308 and Ser473 respectively, and phosphorylation of both sites contributes to the full activation of Akt [40]. In Fig. 1, Akt is somehow phosphorylated at Ser473 even in untreated mice, though both sites of Akt were phosphorylated markedly and immediately in response to PH. This may mean that these two kinases, regulated independently, phosphorylate Akt and contribute to its activation in liver. Though Akt and downstream signals seem to be definitely involved in regulating cell size in liver, the underlying mechanism is still unclear. One possible mechanism may involve the important role of activated Akt (and mTOR) in producing cellular proteins, which leads to hepatocellular hypertrophy [30,41]. In support of this possibility, the serum level of albumin remained within the normal range following PH in LS3-KO mice, whereas it was greatly reduced in control mice (Fig. 9). Activated Akt may have contributed to maintaining high serum albumin levels after PH. The increased protein production by hepatocytes in response to activated Akt might contribute to the responsive increase in cell size. Very interestingly, some mitotic hepatocytes were observed in livers of LS3-KO mice 14 days after PH, though they were not observed in early stage after PH. On the contrary, no mitosis was found in control liver 14 days after PH. Knockout of STAT3 in liver may have led to the extremely delayed response of hepatic mitogenesis after PH,
and Akt-mediated hepatocyte hypertrophy transiently compensated liver mass. Increased cell size in liver of LS3-KO mice 3 days after PH recovered within normal range until 14 days after PH. So it indicates that signalmediated hepatocellular hepertrophy occurred and maintained liver mass until mitosis of hepatocytes starts. Because apoptotic cell death may occur following PH and cause impaired liver regeneration, particularly under certain pathological conditions or as a result of excessive hepatic resection [42–46], and since the anti-apoptotic property of Akt has been widely documented [24–26,47], we investigated the possible involvement of Akt-mediated anti-apoptotic activity in the recovery of liver mass. We evaluated hepatic apoptosis in regenerating liver tissue and found that apoptotic cell death did not increase in control or LS3-KO mice following PH. This finding indicates that Akt contributes to liver regeneration not through its antiapoptotic property, but through other mechanisms. Apoptosis of hepatocytes does not appear to play a central role in maintaining liver regeneration. In conclusion, Akt and downstream signals may play crucial roles in liver regeneration by increasing cell size. Liver regeneration (recovery of liver mass) occurred normally without cell proliferation in LS3-KO mice, but was significantly impaired by inactivation of Akt. Responsive hepatocellular hypertrophy may be indispensable for the recovery of liver mass following hepatectomy under both normal and pathological conditions. Further studies are required to clarify the role of cell proliferation and growth during liver regeneration, and to determine their regulatory mechanisms. The present study, however, may indicate that responsive hepatocellular hypertrophy is more important than cell proliferation for acute liver regeneration.
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