STAT3 Contributes to the Mitogenic Response of Hepatocytes during Liver Regeneration*

STAT3 is rapidly induced during liver regeneration in an interleukin 6 (IL-6)-dependent fashion, and IL-6 is required for normal liver regeneration. We wanted to know whether STAT3 was also required for liver regeneration but disruption of the STAT3 gene during embryonic stages causes lethality. Therefore, an albumin promoter-driven Cre-loxP recombination system was used to create a STAT3 deletion in the adult mouse liver to study the role of STAT3 in liver regeneration. After partial hepatectomy, there was virtually no STAT3 RNA or protein induction in Alb+STAT3 fl/fl livers. STAT3 DNA binding activity was also absent in Alb+ STAT3 fl/fl livers. Unlike in control livers, STAT1 was activated in STAT3 conditional-mutant livers posthepatectomy. Hepatocyte DNA synthesis at 40 h posthepatectomy in Alb+STAT3 fl/fl livers was reduced to approximately one-third of the control. Alb+STAT3 fl/fl livers had abnormalities in immediate-early gene activation that largely correlated with but were not identical to those seen in IL-6−/− livers. G1 phase cyclins including cyclins D1 and E had lower expression levels in Alb+ STAT3 fl/fl livers, indicating an abnormal G1 to S phase transition. Therefore, STAT3 accounts for part of the DNA synthetic response of the hepatocytes during liver regeneration, which cannot be compensated for by induction of STAT1. Normal activation of the MAPK pathway in Alb+STAT3 fl/fl livers reinforces the fact that at least part of the effect of IL-6 on hepatocyte proliferation is not mediated by STAT3. This study provides the first in vivoevidence that STAT3 promotes cell cycle progression and cell proliferation under physiological growth conditions.

Liver regeneration after a two-thirds partial hepatectomy provides an excellent in vivo model for studying cell cycle progression and cell proliferation (1). Differentiated hepatocytes rapidly reenter the cell cycle after surgical removal of the left lateral and median lobes of the liver. Following two rounds of DNA synthesis and cell division, liver mass is restored within 2 weeks (2). Interleukin-6 (IL-6) 1 is known as one of the most important initiators of the regenerative response (3,4). IL-6Ϫ/Ϫ mice have impaired liver regeneration characterized by liver necrosis and failure, a blunted DNA synthetic response in hepatocytes, and reduced gene activation during G 1 phase. Furthermore, STAT3 activation is absent posthepatectomy in IL-6Ϫ/Ϫ livers, which supports a possible pro-proliferative role of STAT3 in hepatocytes (3). IL-6 induces a large spectrum of immediate-early genes (4), in part through direct activation via STAT3 binding sites and in part through cooperative interaction between STAT3 and other transcription factors (5). However, because IL-6 also activates other signaling pathways including the mitogen-activated protein kinase (MAPK) pathway during liver regeneration (4), it is not clear whether the absence of STAT3 is responsible for part or all of the growth defects that are seen in IL-6Ϫ/Ϫ livers.
Signal transducer and activator of transcription 3 (STAT3) belongs to the family of STAT transcription factors which mediate the cellular response to a variety of cytokines and growth factors including IL-6 (6 -8). When IL-6 binds to its specific receptor subunit, it can induce dimerization of the gp130 receptor and activation of the gp130-associated Janus kinase (Jak). The Jaks in turn phosphorylate the specific tyrosines in the intracellular domain of the gp130, providing docking sites for the Src homology 2 (SH2) domain of signaling molecules including STAT3. Once recruited to the receptor chains, STAT3 itself becomes tyrosine phosphorylated by the Jaks, which leads to the dissociation, dimerization, and nuclear translocation of the activated STAT3. Nuclear STAT3 can then bind to specific promoter elements on DNA and activate target gene transcription (9 -11).
Because STAT3 deletion leads to embryonic lethality (12), in vivo data could not be obtained from STAT3 knock-out mice to clarify the role of STAT3 in mediating the effect of IL-6 during liver regeneration. Recently, several laboratories independently have developed conditional STAT3 knock-out in different organs and tissues. Specific disruption of STAT3 in T cells revealed that STAT3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis (13). Also the conditional deletion of STAT3 in the liver demonstrated that STAT3 is essential for the induction of all acute phase response genes downstream of IL-6 (14).
To determine the importance of the STAT3 signaling pathway during liver regeneration and in hepatocyte proliferation, we utilized the STAT3 conditional liver knock-out to study the DNA synthetic response during regeneration. We found that DNA synthesis was partially impaired in Alb ϩ STAT3 fl/fl livers but that liver regeneration still occurred in the absence of STAT3. STAT3 regulates the expression of some IL-6 target genes during liver regeneration but could not account for all of the gene expression abnormalities observed in IL-6Ϫ/Ϫ livers. This is the first in vivo evidence that STAT3 promotes cell cycle progression and cell proliferation by up-regulating immediateearly growth response genes.

MATERIALS AND METHODS
Animal Models-STAT3 fl/fl mice were generated in Dr. Valeria Poli's laboratory as described previously (14). The Alb-Cre transgenic mice were generated by Dr. Christoph Kellendonk (15). STAT3 fl/fl mice were then crossed to the Alb-Cre transgenic mice to generate Alb ϩ STAT3 fl/fl and Alb Ϫ STAT3 fl/fl progenies. Because Alb ϩ STAT3 fl/fl mice are viable and fertile, all conditional knock-out mice and their control littermates used for experiments were generated by mating Alb ϩ STAT3 fl/fl with Alb Ϫ STAT3 fl/fl animals. Genotyping for the Cre transgene was performed by PCR using the following oligonucleotides: CRE1, 5Ј-AG-GCGTTTTCTGAGCATACC-3Ј; CRE 10, 5Ј-TAGCTGGCTGGTGGCA-GATG-3Ј. Two additional oligonucleotides that identify the IL-6 gene were used as an internal control for the PCR reaction: IL-6 Small, 5Ј-CTTCACAGAGGATACCACTCCCAACAG-3Ј; IL-6 No. 4, 5Ј-CTTC-CAGACAGGAAAGGAACCCCTTC-3Ј. The mice were maintained on an ad libitum diet of rodent laboratory chow 5008 (Ralston-Purina Co., St. Louis, MO). Procedures involving animals were conducted with the approval of IACUC and in conformity with the National Institutes of Health guidelines.
Two-thirds Partial Hepatectomy-8 -12-week old mice were anesthetized with isofluorane and subjected to midventral laparotomy, and the left lateral and median lobes were removed (2). Animals were sacrificed at different time points posthepatectomy. Blood was obtained from the heart at the time of harvest, and serum was collected and analyzed by Ani Lytics, Inc. (Gaithersburg, MD).
RNA Isolation, cDNA Microarray, and Northern Blot Analysis-Total RNA was isolated from the remaining liver lobes as described previously (16). 5 g of RNA samples from both the Alb ϩ and Alb Ϫ STAT3 fl/fl livers at 2 h posthepatectomy were reverse-transcribed, labeled with [␣-32 P]dATP, and hybridized to the mouse atlas cDNA expression array as described previously (CLONTECH, Palo Alto, CA). Filters were washed and exposed to autoradiograph film. The intensity of the hybridization signal was analyzed using the ImageQuant program and normalized to the housekeeping genes on the blot. Northern blot analysis was performed using 20 g of total RNA as described previously (16).
Protein Preparation and Western Blot Analysis-Whole liver extracts and nuclear extracts were prepared as described previously (17). Protein electrophoresis, protein transfer, and detection by Western blot and chemiluminescence assay (ECL from Amersham Biosciences, Inc.) were carried out as described previously (18). The primary antibodies Electrophoretic Mobility Shift Assay-10 g of nuclear extracts from liver were used for the electrophoretic mobility shift assay. A doublestranded oligonucleotide from the sis-inducible factor binding element in the c-Fos promoter (5Ј-GATCCTCCAGCATTTCCCGTAAATCCTC-CAG-3Ј) was used for STAT binding as described (19). Supershift experiments were performed by preincubating 1 l of primary antibody with the nuclear extracts at 4°C 2 h before adding the [␣-32 P]ATPlabeled oligonucleotide. Anti-STAT3 (C-20X) and anti-STAT1 (E-23X) antibodies were from Santa Cruz Biotechnology.
Tissue Fixation and BrdUrd Immunohistochemistry-Hepatectomized mice were reanesthetized and subjected to ventral laparotomy. Two hours before harvesting of the liver, animals were injected intraperitoneally with 50 mg of bromodeoxyuridine (BrdUrd)/kg of weight. The remnant liver lobes were removed and fixed immediately in 10% neutral buffered formalin (Formalde-Fresh; Fisher Scientific) for 16 -24 h before the buffer was changed to cold phosphate-buffered saline. The fixed liver was paraffin-embedded, cut to 5 M tissue sections, and adhered to glass slides. BrdUrd immunohistochemical staining was performed on slides as described (18). The avidin-biotin-horseradish peroxidase detection system was from Vector Laboratories (Burlingame, CA; Vectastain Elite ABC and avidin-biotin blocking kit). Anti-BrdUrd antibody (Roche Molecular Biochemicals) was used at a dilution of 1:250. The percentage of BrdUrd-labeled hepatocytes was determined by counting positively stained hepatocyte nuclei in 12 random 20ϫ microscopic fields and calculating the mean. This value was expressed as a fraction of the total number of hepatocytes in a 20ϫ field and averaged 200 cells/field.

RESULTS
Alb-Cre Induced STAT3 Deletion in the Liver-Mice homozygous for the STAT3 floxed allele were generated as described (14). To obtain STAT3 conditional knock-out mice, STAT3 fl/fl mice were bred to a line of Cre recombinase transgenic mice. The albumin promoter was chosen to drive the Cre recombinase to obtain liver-specific deletion of the STAT3 gene. Alb ϩ STAT3 fl/fl mice are fertile, they have no developmental abnormalities, and the gross morphology of the liver is also normal. Therefore, we bred the Alb ϩ STAT3 fl/fl mice to Alb Ϫ STAT3 fl/fl mice and used the F 1 progenies in all of our experiments. Alb Ϫ STAT3 fl/fl littermates were used as controls for the STAT3 conditional-mutants. To confirm that there was little to no STAT3 message or functional protein in the liver posthepatectomy, we performed Northern blot and Western blot analyses. STAT3 mRNA was present in the quiescent Alb Ϫ STAT3 fl/fl liver but not in Alb ϩ STAT3 fl/fl livers. In control livers, the message was rapidly induced after surgery and remained high until 24 h posthepatectomy, whereas there was only minimum induction of the STAT3 mRNA in Alb ϩ STAT3 fl/fl livers (Fig.  1A). We estimated that there was more than 90% deletion of the STAT3 gene. Western blot analyses were used to determine levels of STAT3 protein that had translocated into the nucleus upon activation. STAT3 was not present in the nucleus in quiescent livers, but the level went up rapidly and peaked around 2 h posthepatectomy in Alb Ϫ STAT3 fl/fl livers (Fig. 1B). In contrast, there was no detectable STAT3 protein in Alb ϩ STAT3 fl/fl livers. These results proved that the disruption of STAT3 gene by Cre recombinase in the liver is efficient.
Aberrant STAT1 Activation in Alb ϩ STAT3 fl/fl Livers-A gel mobility shift assay was performed to further confirm that there was no STAT3 DNA binding activity in STAT3 conditional-mutant livers posthepatectomy ( Fig. 2A). Peak STAT3 DNA binding occurred at 2 h posthepatectomy in control livers. The most prominent form of the DNA binding complex was the STAT3/STAT3 homodimer. A low level of STAT3/STAT1 heterodimer as well as an even lower level of STAT1 homodimer band were also observed as we have reported previously (17). There was little induction of the lower two complexes during regeneration in Alb Ϫ STAT3 fl/fl livers. In contrast, there was little STAT3/STAT3 homodimer observed in Alb ϩ STAT3 fl/fl livers after surgery, but a high level of complex was detected migrating at the position of the STAT1 homodimer. Supershift assay was used to confirm the composition of the DNA binding complexes in both the Alb Ϫ and Alb ϩ STAT3 fl/fl livers (Fig. 2B). When preincubated with ␣-STAT3 antibody, the main DNA binding band in Alb Ϫ STAT3 fl/fl livers was supershifted, and multiple supershift complexes were formed. Anti-STAT1 antibody had no effect on this complex, but the putative STAT1/ STAT1 homodimer in Alb ϩ STAT3 fl/fl livers was disrupted by the ␣-STAT1 antibody. ␣-STAT3 antibody had no effect on this complex.
Impaired hepatocyte DNA Synthetic Response in Alb ϩ STAT3 fl/fl Livers Posthepatectomy-Blood was collected from hepatectomized animals and tested for 10 serum indexes. There was no significant difference in the serum indexes from the Alb ϩ compared with the Alb Ϫ STAT3 fl/fl livers, and Alb ϩ STAT3 fl/fl animals regained liver mass normally after 1 week of surgery showing no sign of injury at any time point posthepatectomy (data not shown). We examined the DNA synthetic response in the Alb ϩ and Alb Ϫ STAT3 fl/fl livers using BrdUrd immunohistochemistry. There was a 3-fold difference in the number of hepatocytes undergoing DNA synthesis at 40 h posthepatectomy (p Ͻ 0.02) and reduced DNA synthesis at other time points (Fig. 3, A and B), indicating that Alb ϩ STAT3 fl/fl hepatocytes have an impaired DNA synthetic response during regeneration.
To determine whether disruption of the STAT3 signaling pathway posthepatectomy also has an effect on the MAPK signaling pathway, MAPK activation was examined in the Alb ϩ and Alb Ϫ STAT3 fl/fl hepatectomized livers using an antibody that specifically recognizes phosphorylated Erk1 and Erk2 (Fig. 4). The activation of MAPK in Alb ϩ STAT3 fl/fl livers followed the same kinetics as found in control livers. This experiment was repeated with three different sets of samples. After quantification using scanning densitometry, no consistent or significant difference in the level of either the phosphorylated Erk1 or the phosphorylated Erk2 was observed at any time point. This finding supports the conclusion that STAT3 and MAPK represent two independent IL-6 signaling pathways.
Differential Immediate-early Gene Activation in Alb Ϫ STAT3 fl/fl Livers Versus Alb ϩ STAT3 fl/fl Livers at 2 h Posthepatectomy-By using a mouse cDNA expression array, we successfully identified more than 100 immediate-early genes that are activated at 2 h posthepatectomy, 36% of which also showed IL-6 regulation posthepatectomy. Furthermore, we distinguished the genes that were activated by IL-6 posthepatectomy from those activated by IL-6 injection alone using the same analysis (4). We sought to determine whether the spectrum of genes that are regulated by STAT3 posthepatectomy overlaps with those activated by IL-6. Total RNA was isolated from the control and STAT3 conditional-mutant livers at 2 h posthepatectomy and used for the microarray analysis as described previously (Fig. 5). A smaller number of immediateearly genes were differentially expressed in Alb Ϫ STAT3 fl/fl livers versus Alb ϩ STAT3 fl/fl livers compared with those differentially expressed in the IL-6ϩ/ϩ and Ϫ/Ϫ livers. However, the degree of overlap between the two gene sets was high. Only 1 of 10 STAT3 target genes had not previously been identified as an IL-6 target gene (lymphotoxin receptor). Of the nine IL-6 target genes, six were differentially expressed in the IL-6ϩ/ϩ and Ϫ/Ϫ livers posthepatectomy, including c-myc, TNFR2, GADD-45, serine protease inhibitor 2.4, c-fos, and Egr-1. Three did not show IL-6 regulation posthepatectomy but were activated by IL-6 injection in the liver (TDAG51, membrane glycoprotein gp130, and Erp72, the endoplasmic reticulum stress protein) (4). Interestingly, Egr-1, which was down-regulated in IL-6Ϫ/Ϫ livers 2 h posthepatectomy, was up-regulated in Alb ϩ STAT3 fl/fl livers, indicating a STAT3-independent activation of Egr-1 posthepatectomy.
Representative IL-6 Target Genes Regulated by STAT3 Posthepatectomy-In IL-6Ϫ/Ϫ livers, abnormalities in the induction and temporal expression of several important immediate- To determine whether STAT3 is responsible for activation of these IL-6-regulated genes, we performed Northern analysis using RNA samples isolated from both the Alb ϩ and Alb Ϫ STAT3 fl/fl livers at different time points posthepatectomy (Fig.  6A). As in IL-6Ϫ/Ϫ livers, c-fos, and junB expression were significantly reduced in Alb ϩ STAT3 fl/fl livers at all time points compared with Alb Ϫ STAT3 fl/fl livers. Peak activation of c-myc was delayed in Alb ϩ STAT3 fl/fl livers compared with Alb Ϫ STAT3 fl/fl livers. IGFBP-1 is one of the most induced immediate-early genes during liver regeneration. Its activation is in part regulated by IL-6 via direct interaction between STAT3, AP-1, and HNF-1 transcription factors (5). In STAT3 condition-al-mutant livers, IGFBP-1 expression is reduced at 2 h posthepatectomy by 2-fold based on the result of three different sets of samples. However, the change is not as great as that seen in the IL-6Ϫ/Ϫ livers (5). Interestingly, Egr-1 is expressed at a lower level at 2 h posthepatectomy in IL-6Ϫ/Ϫ livers but a higher level at all time points in Alb ϩ STAT3 fl/fl livers compared with Alb Ϫ STAT3 fl/fl livers. We examined the nuclear extracts from the Alb ϩ and Alb Ϫ STAT3 fl/fl livers representing early time points posthepatectomy to see whether the c-Fos protein level was reduced in Alb ϩ STAT3 fl/fl livers as seen in IL-6Ϫ/Ϫ livers (Fig. 6B). In Alb Ϫ STAT3 fl/fl livers, induction of c-Fos was detected as early as 1 h, and there was a peak induction at 2 h posthepatectomy. There was little induction of c-Fos until 4 h posthepatectomy in Alb ϩ STAT3 fl/fl livers. We also examined the level of activated STAT1 protein posthepatectomy using nuclear extracts from the STAT3 conditionalmutant and control livers. The STAT1 protein level was markedly induced in the nucleus of Alb ϩ STAT3 fl/fl livers, whereas in control livers there was only a slight increase in the STAT1 level posthepatectomy. This result correlated with the increased STAT1 DNA binding activity in Alb ϩ STAT3 fl/fl livers posthepatectomy.
Partially Impaired Activation of the G 1 Phase Cyclins in Alb ϩ STAT3 fl/fl Livers-IL-6Ϫ/Ϫ livers have defects in G 1 /S phase transition as manifested by a depressed cyclin D1 expression during regeneration. To understand if STAT3 also plays a role in promoting the G 1 to S phase transition of the hepatocytes, we sought to determine whether there is abnormal expression of cell cycle regulatory proteins in Alb ϩ STAT3 fl/fl livers, especially those expressed during G 1 phase progression and at the G 1 /S transition (3). We examined the protein level of cyclin D1 as well as another G 1 phase cyclin, cyclin E, using Western blot analysis (Fig. 7). Cyclin D1 expression was normal at earlier time points during the cell cycle but was reduced at the peak of DNA synthesis (40 h posthepatectomy) and remained low at all later time points in the Alb ϩ compared with Alb Ϫ STAT3 fl/fl livers. This observation correlated with that made in IL-6Ϫ/Ϫ livers. Cyclin E was activated as early as 16 h posthepatectomy in Alb Ϫ STAT3 fl/fl livers and peaked around 48 h posthepatectomy. In Alb ϩ STAT3 fl/fl livers, the induction of cyclin E was delayed until 32 h posthepatectomy, and the level of expression was lower at later time points compared with that in control livers. The upper band observed in the cyclin E blot (Fig. 7) was most likely the phosphorylated and inactive form of cyclin E. It is unknown whether cyclin E is regulated by IL-6 during liver regeneration because the expression of cyclin E posthepatectomy was not examined in IL-6Ϫ/Ϫ livers. DISCUSSION Evidence suggests a link between STAT activation and cellular proliferation (20 -25). A number of transformed cell lines as well as samples from human cancers are reported to contain constitutively activated STAT3. Furthermore, a constitutively active form of STAT3 causes cellular transformation in immo-bilized fibroblasts (26). The study reported here is the first in vivo evidence that STAT3 can directly participate in cell cycle progression. STAT3 was required for the activation of several immediate-early genes at the gene expression level including c-fos and junB. These two genes are the most strongly affected immediate-early genes in IL-6Ϫ/Ϫ livers, and their expression is likely to be directly regulated by STAT3 because their full transactivation requires the STAT binding elements in their promoters (19,27). STAT3 was also necessary for the activation of G 1 phase cyclins that support DNA synthesis and mitosis. DNA synthesis posthepatectomy was partially impaired in the absence of STAT3, although mass restoration occurred as in IL-6Ϫ/Ϫ livers (3). Regaining liver mass despite deficient DNA synthesis is a general phenomenon in animal models that are defective in liver regeneration, which most likely results from hypertrophy of hepatocytes despite G 1 /S arrest (3, 18, 28 -30). The results obtained from Alb ϩ STAT3 fl/fl are important because they represent the first example of a pro-proliferative role of STAT3 under physiological growth conditions in vivo.
The STAT family consists of seven family members, and in vitro they bind to a similar consensus element on the DNA, TTCnnn(n)GAA (31)(32)(33). From the knock-out mouse models now available, only STAT3 plays an essential role in early embryonic development (12). All of the other STAT knock-outs have relatively mild phenotypes correlated with rather specific defects in response to certain cytokines or growth factors (12, 34 -37). Although they bind to a similar DNA binding site, STATs may interact with co-activator or co-repressor to regulate different sets of their own downstream targets (5, 38 -44). Furthermore, the tissue-specific distribution of some STATs may also contribute to their functional specificity (45). The STAT1 knock-out has a specific defect in the interferon-␥ response (46). STAT1 is the only STAT in addition to STAT3 that is activated during liver regeneration (17). In the absence of STAT3, STAT1 activation was dramatically increased posthepatectomy, suggesting that normally STAT3 may inhibit STAT1 activation during liver regeneration by unknown mechanisms. In the STAT3 conditional knock-out, we observed that the spectrum of genes in which expression levels are reduced is smaller compared with that seen in the IL-6 knock-out. There FIG. 5. Immediate-early genes differentially expressed in Alb ؊ versus Alb ؉ STAT3 fl/fl livers at 2 h posthepatectomy. 5 g of total RNA samples isolated from the Alb Ϫ and Alb ϩ STAT3 fl/fl livers at 2 h posthepatectomy were reverse-transcribed and hybridized to a mouse cDNA expression array as described previously (4). Immediate-early genes that were differentially expressed in Alb Ϫ STAT3 fl/fl livers versus Alb ϩ STAT3 fl/fl livers are summarized in this graph. The solid columns represent previously identified IL-6 target genes. Results were quantified using the Image-Quant program and normalized to the housekeeping genes on the array. Differential expression greater than 3-fold is shown. could be two reasons for this observation. One is that MAPK contributes to the immediate-early gene induction during liver regeneration. Second, the expression of a subset of STAT3 target genes in the liver might be compensated for by STAT1. The expression of IGFBP-1 is activated by STAT3 in coordination with AP-1 and HNF-1 transcription factors via an HNF-1 binding site during liver regeneration. Its expression level is 2-fold lower in Alb ϩ STAT3 fl/fl livers than control livers compared with more than 3-fold lower in IL-6Ϫ/Ϫ livers. It represents a class of STAT3 target genes for which expression may be rescued partially in the presence of STAT1, but further analysis would be required to determine whether STAT1 is able to substitute for STAT3 in binding HNF-1. However, STAT1 activation was not sufficient to compensate for all STAT3 function because disruption of STAT3 still led to downregulation of several immediate-early genes, and the level of the reduction was comparable with that seen in the IL-6 knockouts. This is consistent with the previous report that the STATbinding element in the JunB promoter does not bind or respond to STAT1 (27).  It was demonstrated clearly that activation of the MAPK pathway is a key signal for the G 1 phase progression of the proliferating hepatocytes after partial hepatectomy (47). There are two peaks of MAPK activation during regeneration, one at early G 1 and the other during mid-late G 1 phase. In rat liver, the second peak of activation occurs around 10.5 h, whereas in mouse liver it is around 24 h posthepatectomy, corresponding to a later S phase onset in the mouse liver. In IL-6Ϫ/Ϫ livers, the first peak of MAPK activation is delayed until 12 h compared with 2 h posthepatectomy in IL-6ϩ/ϩ livers; the factor responsible for this activation in the absence of IL-6 is not known (4). In contrast, MAPK activation is normal in Alb ϩ STAT3 fl/fl livers during the early time points posthepatectomy, suggesting that IL-6 activation of the MAPK pathway is independent of STAT3 activation. On the other hand, it also suggests that the STAT3 effect on hepatocyte DNA synthesis and cell proliferation is not secondary to direct effects on the MAPK pathway. Interestingly, at the peak of S phase, which is approximately 40 h posthepatectomy in mouse livers, the percentage of hepatocytes undergoing DNA synthesis is 5-fold lower in IL-6Ϫ/Ϫ livers than IL-6ϩ/ϩ livers, whereas the difference between the Alb ϩ and Alb Ϫ STAT3 fl/fl livers is smaller at about 3-fold. This could be explained by the contribution of the MAPK pathway to hepatocyte proliferation.
A certain percentage of IL-6Ϫ/Ϫ animals die after surgery because of liver necrosis and failure (3), whereas Alb ϩ STAT3 fl/fl animals were viable after hepatectomy with no signs of liver injury. It is possible that the induction of STAT1 in Alb ϩ STAT3 fl/fl livers can rescue some of the defects caused by lack of STAT3 induction. The MAPK pathway may also play a role in reducing injury and cell death in the liver. Furthermore, there could be other signaling pathways activated by IL-6 that are important for maintaining normal liver cell function during liver re-growth that still await characterization.