INTRODUCTION

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The mammalian liver is one of the few adult organs capable of completely regenerating itself in response to cellular injury from toxins, viral infections, or tissue removal (15, 38, 46). Liver regeneration after two-thirds partial hepatectomy (PH) represents a balance between hepatocyte proliferation and the maintenance of hepatocyte-specific gene expression required for liver homeostasis (22, 46). A potent activation of hepatocyte immediate early transcription factors is observed during liver regeneration and includes c-Jun, c-Fos, c-Myc, NF-B, signal transducers, and activators of transcription 3 (stat3) and the CCAAT/enhancer protein  (C/EBP) genes (7, 9, 25, 46). Furthermore, maintenance of hepatocyte-specific gene transcription is coincident with sustained expression of hepatocyte nuclear factor genes (16, 20, 41). More recent genetic data demonstrated that the cytokine interleukin-6 (IL-6) plays an important role in establishing responsiveness of hepatocytes to growth factors which are released after liver injury (8, 54). In a PH model of liver regeneration, homozygous null interleukin-6 (IL-6) or type 1 tumor necrosis factor receptor (TNFR-I) mice exhibited a 70% reduction in hepatocyte replication and this proliferation defect was eliminated by an intraperitoneal injection of IL-6 prior to surgery (8, 43, 54). This proliferation defect was accompanied by a failure to induce the normal expression pattern of immediate early transcription factors, including stat3, AP-1, NF-B, and c-myc.

Genetic studies with transgenic and knockout mice have implicated several growth-regulated transcription factors in mediating hepatocyte proliferation during liver regeneration. After PH, albumin promoter driven c-myc transgenic mice exhibited a 10-h-earlier onset of hepatocyte proliferation, which correlated with premature expression of cyclin A and cdc2 genes (14). The C/EBP transcription factors have been shown to be pivotal regulators of energy metabolism, cellular differentiation and proliferation (10, 11, 40). Liver regeneration elicits diminished expression of the antiproliferative C/EBP isoform, while inducing a compensatory increase in the expression of the C/EBP and C/EBP genes (13, 32, 46). Use of C/EBP-deficient mice in PH experiments reveals a 75% reduction in replicating hepatocytes with coincident decreases in expression of immediate-early EGR-1 transcription factor and in cyclin B and E expression levels (21). More recent regeneration studies with cAMP-responsive promoter element modulator (crem)-deficient mice demonstrate a 50% reduction in hepatocyte DNA replication, which is delayed by 10 h after PH and paralleled by diminished expression of c-fos, as well as the cell cycle regulators cyclin A, B, D, E, and cdc2. (44). These studies demonstrate that the induction of immediate-early transcription factors is essential for hepatocyte proliferation after PH, but transcriptional mechanisms mediating the later stages of hepatocyte cell cycle progression are not completely understood.

Rodent HNF-3 (5, 30, 31) and Drosophila homeotic fork head proteins (51) were the first identified members of an extensive family of transcription factors which share homology in the winged helix DNA-binding domain (3). The winged helix DNA motif consists of a 100-amino-acid region that conforms to a modified helix-turn-helix motif and binds to DNA as a monomer (3). The winged helix family consists of over 50 transcription factors which play important roles in cellular differentiation and organ morphogenesis (26, 39), in cellular proliferation and transformation (17, 18, 33, 34), and in regulating expression of apoptosis-promoting genes (2). The HNF-3/fork head homolog-11 (HFH-11; also known as Trident and Win, but is distinct from the winged helix nude gene [39]) is a proliferation-specific member of the winged helix transcription factor family. Previous studies have shown that HFH-11/Trident is expressed in every tumor-derived epithelial cell line examined and that it is induced by serum prior to the G₁/S transition (27, 28, 56, 57). In situ hybridization studies demonstrate that HFH-11 expression is observed in proliferating cells of 16-day-postcoitum mouse embryos, including the liver, intestine, lung, and renal pelvis (57). In adult organs HFH-11 expression is extinguished in the postmitotic, differentiated cells of the liver and lung, but its expression continues in proliferating cells of adult tissue, primarily in the thymus, testis, small intestine, and colon (57). We demonstrated that, during liver regeneration, HFH-11 expression is reactivated prior to hepatocyte DNA replication and thus exhibits the induction kinetics of a delayed-early transcription factor (57). Furthermore, perinatal lethality of hfh11/trident-deficient mice is accompanied by an abnormal polyploid phenotype in embryonic hepatocytes and cardiomyocytes (29). These results suggest that the hfh11/trident gene functions in these embryonic cells to prevent multiple rounds of DNA replication prior to the completion of mitosis.

To examine whether HFH-11 regulates cell cycle progression during liver regeneration, transgenic mice were created in which ectopic hepatocyte expression of the transcriptionally active HFH-11B isoform occurred. Liver regeneration studies with these mice demonstrate that premature HFH-11 expression caused an 8-h acceleration in the onset of hepatocyte DNA replication and mitosis. This early proliferation of regenerating transgenic hepatocytes is consistent with the premature induction of genes involved in promoting DNA replication, DNA repair, and mitosis. Furthermore, in nonregenerating transgenic livers, abnormal hepatocyte proliferation was not observed because the HFH-11B transgene protein was retained in the cytoplasm. We found that mitogenic signalling induced during liver regeneration is required for nuclear localization of the HFH-11B transgene protein and that its premature availability in regenerating transgenic liver allowed nuclear translocation to occur 8 h earlier than would otherwise have been the case.

	MATERIALS AND METHODS

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Generation of TTR-HFH-11B transgenic mice. The transthyretin (TTR) minigene construct (Fig. 2) consists of the 3-kb TTR promoter region, the first and second TTR exons fused to the simian virus 40 (SV40) 3' end and poly(A) sequences (6, 55). The ATG sequence in the TTR first exon is mutated and allows insertion of the HFH-11B cDNA in the StuI site of the TTR second exon. This TTR transgene therefore provides a 5' splice site for more reliable expression in transgenic mice (53). The 2.6-kb human HFH-11B cDNA was excised with EcoRI-HindIII, blunt ended with Klenow fragment of DNA polymerase I, and ligated into a unique StuI site located in the second exon of the pTTR-exv3 minigene vector (see Fig. 2). The 7.0-kb HindIII fragment containing the 3-kb TTR promoter driving HFH-11B cDNA expression was separated from the vector fragment by regular agarose gel electrophoresis (FMC) and then purified from the agarose by using the GeneClean II kit (Bio 101, Inc.). The transgene was injected into the pronuclei of CD1 mouse eggs at a concentration of 3 ng/µl, and transgenic mice were generated as described by Hogan et al. (24). After microinjection of the TTR-HFH-11B transgene construct into CD-1 fertilized mouse eggs and transfer to surrogate mothers, 61 mice were born and 3 of these carried the transgene as identified by PCR. Identification of mice carrying the HFH-11B transgene was performed by PCR analysis of genomic DNA extracted from the tails of 2- to 4-week-old mice. The primers used were 5'-AAAGTCCTGGATGCTGTCCGAG-3' (sense TTR exon two 5' primer) and 5'-CAGACATGATAAGATACATTGATG-3' (antisense SV40 3' primer). The 5' and 3' primers for an internal control thyroid stimulating hormone gene were 5'-TCC TCA AAG ATG CTC ATT AG-3' and 5'-GTA ACT CAC TCA TGC AAA GT-3'.

Surgical procedure for mouse partial hepatectomy. For liver regeneration studies, 10- to 12-week-old male CD-1 mice (30 to 35 g [body weight]) were anesthetized with methoxyflurane (Metofane; Schering-Plough Animal Health Corp., Union, N.J.) and subjected to midventral laparotomy with a two-third liver resection (left lateral, left median, and right median lobes) (23). Extra care was taken to avoid excision of the gallbladder situated at the cleft between the left and right median lobes. One subcutaneous injection of ampicillin (50 µg/g [body weight]) in saline was given to the animal after the surgical procedure. Two hours before the remnant liver was harvested, animals were injected intraperitoneally with 5-bromo-2'-deoxyuridine (BrdU; 50 µg/g [body weight]; 0.2% solution in phosphate-buffered saline [PBS]). Four to six mice from each group were sacrificed at 4-h intervals from 24 to 52 h and at 68 h posthepatectomy by CO₂ asphyxiation. A portion of liver tissue was used to prepare total RNA; another portion was fixed overnight in 4% paraformaldehyde (in PBS solution at pH 7.4) at 4°C, paraffin embedded the second day, and sectioned as 6- to 8-µm sections by using a microtome. The sectioned tissues were used for (i) routine microscopy with hematoxylin and eosin stain, (ii) evaluation of the fraction of hepatocytes undergoing DNA synthesis by BrdU incorporation, (iii) in situ hybridization with antisense ³³P-labeled riboprobes, and (iv) immunohistochemistry analyses with HFH-11 antibody (57).

RNA extraction and RNase One protection assay. Total RNA was extracted from mouse liver by an acid guanidium thiocyanate-phenol-chloroform extraction method with RNA-STAT-60 (Tel-Test "B" Inc., Friendswood, Tex.). The HFH-11B antisense RNA probe was generated from the PCR-derived BamHI-EcoRI HFH-11A cDNA pGEM1 clone (409-bp fragment) that contained exon AII and sequences encoding the 58 amino acids N-terminal to exon AII (amino acids 366 to 469) as described previously (57). The TTR transgene probe was made from an EcoRI-digested pTTRExV3 template DNA with SP6 RNA polymerase (55) and detects expression of both the endogenous TTR and the transgene. The protected fragments of TTR and transgene were 90 and 310 nucleotides, respectively. The C/EBP antisense RNA probe was made from an EcoRI-digested rat C/EBP cDNA pGEM-1 template (867 to 1,392 bp) with SP6 RNA polymerase. pTRI vector containing cDNA templates for mouse p34cdc2, cyclin B1, and cyclophilin were purchased from Ambio, Inc. (Austin, Tex.). The protected fragments of p34cdc2, cyclin B1, and cyclophilin were 250, 214, and 113 nucleotides, respectively.

Immunohistochemistry and in situ hybridization. Livers from BrdU-injected mice (50 µg/g [body weight]; 2 h before harvesting) were fixed in 4% paraformaldehyde overnight, embedded in paraffin, prepared for histological analysis, and immunohistochemically stained with an anti-BrdU monoclonal antibody according to the manufacturer's protocol (Boehringer Mannheim) or with the HFH-11-specific antibody (57). For immunohistochemistry, the paraffin wax was removed from sections with xylenes, after which they were rehydrated in ethanol and then placed in PBS plus 0.25% Triton X-100 (PBT). We used a microwave-based antigen retrieval method to enhance the antigenic reactivity of antibodies with paraformaldehyde-fixed dewaxed paraffin-embedded sections as described previously (59). Primary antibodies were detected by using secondary anti-mouse immunoglobulin G coupled to horseradish peroxidase staining with the appropriate substrates (Vector Laboratories). To detect nuclear localization of the HFH-11 protein during mouse liver regeneration, we used the HFH-11 antibody (57) for immunohistochemistry staining of liver sections (see Fig. 2) with AEC as the peroxidase substrate (stains red), followed by counterstaining with hematoxylin (stains nuclei blue). The number of BrdU-positive hepatocytes undergoing DNA synthesis was determined by randomly counting the positive BrdU staining nuclei per 1,000 in a total of at least 3,000 hepatocytes. Mitotic figures in hepatocytes were counted and quantitated as a percentage of at least a total of 1,500 cells under 10 high-power fields at the indicated time after hepatectomy. The data from BrdU labeling experiments, mitotic figures, and fold induction of cdc2 expression levels are represented as the means ± the standard error of the mean. The Student t test was used to compare the different parameters between the two groups by using the Analysis ToolPak in Microsoft Excel 98. A P value of <0.05 was considered significant.

Mouse cDNA array analysis. RNA samples were isolated from liver tissues at 24, 28, 32, and 40 h posthepatectomy. ³²P-labeled cDNA probe was prepared by using mouse liver poly(A)⁺ RNA from wild-type and transgenic animals according to the user manual (Clontech, Palo Alto, Calif.). The labeled cDNA was hybridized to Atlas Mouse cDNA array membranes at 65°C overnight, and the blots were washed in 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 65°C. The arrays were then exposed to phosphor screens overnight and scanned with a Storm 860 PhosphorImager. Quantitation of expression levels was determined by utilizing the ImageQuant analysis program. All measurements were stored in a computer database for analysis and interpretation by using Microsoft Excel 98. Each array blot contained 588 well-characterized genes, such as cytokines and their receptors, transcription factors, apoptosis-related genes, and cell cycle regulators. Each blot also contained nine housekeeping genes for normalizing the hybridization signals.

	RESULTS

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Induced expression and nuclear translocation of HFH-11 occurs prior to DNA replication in regenerating mouse hepatocytes at 32 h post-PH. Previous studies of regenerating rat liver demonstrated that induction of HFH-11 expression precedes hepatocyte entry into DNA replication (S phase), but it follows immediate-early gene expression at 4 h post-PH (57). HFH-11 thus exhibits the expression kinetics of a delayed-early transcription factor gene. In regenerating mouse liver, DNA replication initiates at 40 h post-PH and is delayed compared to regenerating rat liver. In order to determine the expression pattern of HFH-11 during mouse liver regeneration, we performed in situ hybridization of paraffin sections of regenerating mouse liver by using ³³P-labeled antisense HFH-11 RNA probe. In regenerating mouse liver, HFH-11 hybridization signals are barely visible by 24 h after PH (Fig. 1A and B), and its expression increases to maximal levels by 32 h post-PH (Fig. 1C to F). Furthermore, we observed more intense HFH-11 labeling in regenerating hepatocytes surrounding the periportal region (Fig. 1C to F), which are the first hepatocytes to initiate DNA replication during liver regeneration (19, 50). To detect nuclear localization of the HFH-11 protein during mouse liver regeneration, we used the HFH-11 antibody (57) for immunohistochemistry staining of liver sections (Fig. 2) with AEC as the substrate (stains red), followed by hematoxylin counterstaining (stains nuclei blue). The nuclear localization studies reveal cytoplasmic HFH-11 protein staining at 24 h post-PH (Fig. 2A and B), perinuclear HFH-11 staining by 28 h post-PH (Fig. 2C) and, ultimately, nuclear translocation of the HFH-11 protein is observed by 32 h after PH (Fig. 2D to E, red nuclei). These expression studies of regenerating mouse liver indicate that nuclear translocation of the HFH-11 protein occurs during the G₁-to-S-phase transition.

View larger version (331K): [in this window] [in a new window]   FIG. 1.   Induction of HFH-11 expression in regenerating mouse liver precedes DNA replication and is localized to hepatocytes of the periportal region. To determine the temporal and spatial expression pattern of HFH-11, paraffin sections of regenerating wild-type (WT) mouse liver were prepared at various times (hours) after PH and used for in situ hybridization with 33P-labeled antisense HFH-11 RNA probe. Shown in the left panels are the bright-field illuminations and in the right panels are the dark-field illuminations depicting the hybridization signals. Indicated on the liver sections are the portal vein (PV), the periportal region (PP) comprising the hepatocytes surrounding the portal vein, and the central vein (CV). In regenerating mouse liver, HFH-11 expression initiates at 24 h post-PH (A and B) and is maximally expressed at 32 h post-PH (C and D), exhibiting more-intense HFH-11 labeling in hepatocytes of the periportal region. (E and F) The HFH-11 periportal expression pattern continues in regenerating mouse liver at 40 h post-PH. (G and H) Nonregenerating hepatocytes from the transgenic (TG) T38 line (see Fig. 2) reveal ectopic HFH-11B expression throughout the liver parenchyma, but HFH-11 hybridization signals are more abundant in the periportal region. (I and J) Expression of the HFH-11 transgene continues in replicating hepatocytes at 34 h post-PH. Note that the in situ hybridization for regenerating wild-type liver is exposed for 1 week, which is three times longer than for the transgenic mouse livers.

View larger version (150K): [in this window] [in a new window]   FIG. 2.   Hepatocyte nuclear localization of HFH-11 protein in regenerating wild-type and transgenic livers. To determine nuclear localization of the HFH-11 protein, paraffin sections of regenerating wild-type (WT; A to E) or transgenic (TG; F to K) mouse livers were prepared at various times (hours) after PH and used for immunohistochemical staining with the HFH-11 antibody (57). The HFH-11 antibody was detected with the horseradish peroxidase AEC substrate (stains red), followed by hematoxylin counterstaining (stains nuclei blue). (A to D) Nuclear localization of the HFH-11 protein in regenerating wild-type hepatocytes is observed at 32 and 36 h post-PH (red nuclei [indicated by arrows]), while perinuclear staining is found at 28 h post-PH, and their nuclei are counterstained blue by hematoxylin (indicated by asterisks). (E to I) Hepatocyte nuclear localization of the HFH-11B transgene protein requires mitotic signalling induced during liver regeneration. HFH-11B transgene protein is diffusely distributed in the cytoplasm of nonregenerating transgenic hepatocytes, and hematoxylin counterstains the nuclei blue (F [indicated by an asterisk]). In regenerating transgenic hepatocytes, HFH-11B nuclear localization (red, indicated by arrows) is observed at 24, 28, 32, and 36 h post-PH (H and I), while perinuclear and nuclear staining is found at 16 h post-PH (G). Hepatocyte nuclear translocation of the HFH-11B protein therefore occurs 8 h earlier in regenerating transgenic liver.

Hepatocyte nuclear localization of the HFH-11B transgene protein requires mitotic signalling induced during liver regeneration. In order to ectopically express the transcriptionally active human HFH-11B isoform in hepatocytes (57), we generated transgenic mice by using the 3-kb TTR minigene construct (Fig. 3A), which effectively targets transgene expression to hepatocytes (53, 55). Three founder transgenic mice were obtained (T38, T69, and T70) that expressed the HFH-11B transgene at different levels (Fig. 3B). None of the transgenic lines exhibited aberrant hepatocyte proliferation or defects in the liver architecture, nor was there a significant difference in liver/body weight ratio (data not shown). Furthermore, liver function was not affected in the TTR-HFH-11B transgenic mice as assessed by determining the normal serum levels of glucose, alkaline phosphatase, albumin, liver aminotransferase enzymes, and bilirubin (Table 1). We used the T38 TTR-HFH-11B transgenic line for further liver regeneration studies because it displayed the highest ectopic expression of the HFH-11B transgene (Fig. 3) and its expression was observed throughout the parenchyma of nonregenerating transgenic liver, albeit, it exhibited a more intense periportal expression (Fig. 1G and H). Sustained expression of the HFH-11B transgene was also observed throughout the parenchymal cells in regenerating livers of transgenic mice (Fig. 1I and J).

View larger version (52K): [in this window] [in a new window]   FIG. 3.   The 3-kb TTR promoter directs HFH-11B transgene expression in the adult liver. (A) Diagrammatical representation of the mouse 3-kb TTR promoter-HFH-11B transgene construct. Transgenic mice were created with the 3-kb TTR promoter region (small open box) driving expression of the human HFH-11B cDNA (striped box), which was cloned into the TTR second exon (large open box) that contains the SV40 polyadenylation signal (black box) (5, 6, 55). (B) Analysis of HFH-11B transgene expression in transgenic mouse livers. Total liver RNA was isolated from F1 transgenic mouse lines T38 (lanes 1 and 18), T60 (lanes 25 and 30), and T70 (lanes 37 and 38) and nontransgenic litter mates (lanes 10, 17, 26, and 35) and used for RNase protection assays with either the TTR-SV40 transgene or HFH-11B antisense-labeled RNA probes (see Materials and Methods). As reported previously, expression of the transgene produces RNase-resistant 310 nucleotide product (HFH-11B transgene), whereas expression of the endogenous TTR gene elicits an RNase protected fragment of 90 nucleotides (6, 55). Note that RNase protection with adult liver RNA and the HFH-11B probe only detected HFH-11B expression in the transgenic mouse lines. Lane numbers correspond to mouse numbers in each transgenic line.

Premature hepatocyte expression of HFH-11B accelerates the onset of DNA synthesis and mitosis in regenerating transgenic mouse liver. To determine whether earlier nuclear translocation of the HFH-11B protein in regenerating liver would alter the timing of hepatocyte DNA replication, liver regeneration studies were performed with wild-type and TTR-HFH-11B transgenic mice, and DNA synthesis was monitored by immunohistochemical staining of BrdU incorporation into DNA (8, 54). We used three to six regenerating livers per time point, and the results of the BrdU labeling studies are shown graphically in Fig. 4G. Consistent with previous reports (8, 14, 21, 44, 54), only a few of the wild-type hepatocytes exhibit BrdU incorporation at 36 h post-PH, and hepatocyte replication reaches a maximum by 40 h and is diminished by 48 h after surgery (Fig. 4A, C, and E). In contrast, maximal hepatocyte DNA replication in regenerating transgenic mouse liver occurs at 32 h post-PH (Fig. 4B, D, F, and G), a result consistent with an 8-h-earlier nuclear translocation of the HFH-11B transgene protein (Fig. 2). Consistent with regenerating wild-type livers (19, 50), the initiation of hepatocyte proliferation is restricted to the periportal region of regenerating transgenic livers but becomes more diffuse at later time points (Fig. 4D to F). Furthermore, initiation of mitosis occurred 8 h earlier in regenerating transgenic hepatocytes, as evidenced by an increase in the number of mitotic bodies present at 40 h post-PH (Fig. 4H). The total numbers of hepatocytes undergoing DNA synthesis and mitosis, however, were not significantly different between transgenic and wild-type regenerating livers (Fig. 4G and H). These studies suggest that earlier nuclear localization of the HFH-11B protein accelerates the timing of hepatocyte entry into DNA replication and mitosis.

View larger version (211K): [in this window] [in a new window]   FIG. 4.   Premature hepatocyte expression of HFH-11B accelerates DNA synthesis in regenerating transgenic mouse liver. Wild-type and TTR-HFH-11B transgenic mice were subjected to two-thirds PH and, 2 h prior to harvesting the regenerating livers, the mice received an intraperitoneal injection of BrdU to be detected by a monoclonal antibody to BrdU (see Materials and Methods). Immunohistochemical detection of hepatocytes incorporating BrdU is shown with regenerating wild-type (left panels) and transgenic (right panels) mouse livers. Maximal hepatocyte DNA replication for regenerating wild-type liver occurs at 40 h post-PH, whereas the peak of transgenic hepatocyte replication is observed 8 h earlier at 32 h post-PH. Note that wild-type regenerating livers also initiate DNA replication in hepatocytes surrounding the periportal region. Comparable BrdU incorporation is observed in regenerating wild-type and transgenic livers at 48 (E) and 40 (F) h post-PH, respectively. (G) Kinetics of BrdU incorporation into hepatocyte DNA during wild-type (WT) and transgenic (Tg) liver regeneration between 24 and 68 h post-PH. BrdU-positive hepatocytes (per 1,000 nuclei) from each sample were counted among at least 3,000 total hepatocytes, and the mean and standard deviation for each time point was calculated by using three to six regenerating livers. In regenerating transgenic livers, we observed an 8-h-earlier peak in hepatocyte DNA replication and no increase in the total number of replicating hepatocytes. An additional BrdU labeling time point (34 h post-PH) was used for regenerating transgenic liver. (H) Kinetics of hepatocyte mitosis during wild-type and transgenic liver regeneration between 32 and 68 h post-PH. Hepatocyte mitotic figures were counted in 10 high-power fields at the indicated time post-PH and are presented as a percentage of the total number of hepatocytes. The means from three regenerating mouse livers with the corresponding standard deviations are shown.

Earlier expression of DNA repair enzymes and cyclin genes in regenerating TTR-HFH-11B transgenic mouse livers. To identify hepatocyte proliferation genes that are differentially expressed in regenerating transgenic and wild-type mouse livers, Atlas Expression cDNA Array Blots (Clontech) were hybridized with radioactive cDNA prepared from various stages of regenerating mouse liver (24, 32, and 40) h post-PH; see Materials and Methods). The Atlas Expression cDNA Array Blot contains 588 distinct cDNAs spotted in duplicate and organized in quadrants containing genes participating in similar cellular pathways. In regenerating transgenic mouse liver, a threefold increase in expression of DNA repair genes was observed by 24 h post-PH (Fig. 5, cDNAs 1 to 3). This level of increase is normally induced during hepatocyte DNA replication at 40 h post-PH. These DNA repair genes included the double-stranded break repair genes XRCC1 and mHR21spA (mouse homolog of yeast rad21 gene) genes and the nucleotide excision repair mHR23B (mouse homolog of yeast rad23 gene) gene. The earlier induction of DNA repair genes in the TTR-HFH-11B regenerating livers is consistent with the accelerated hepatocyte DNA replication phenotype.

View larger version (81K): [in this window] [in a new window]   FIG. 5.   Earlier expression of DNA repair and cyclin genes in regenerating transgenic livers. Radioactive cDNA was prepared from regenerating wild-type and transgenic livers at 24, 32, and 40 h post-PH and hybridized to six distinct Atlas mouse cDNA expression array blots. Composite images of two columns comparing hybridization signals of the DNA repair (A) or cyclin (B) genes in regenerating wild-type (WT) or transgenic (Tg) livers at the indicated hours post-PH are shown. Blots are normalized to the nine housekeeping cDNAs prior to comparing signals of other cDNAs which are spotted in duplicate on the blot. Arrows indicate transgenic regenerating cDNA signals which are increased compared to regenerating wild-type livers, and quantitation of the cDNA hybridization signals allowed determination of the fold induction. The numbering of cDNAs are as follows: 1, double-stranded break repair gene XRCC1; 2, mHR21spA (mouse homolog of the yeast rad21 gene); 3, the nucleotide excision repair mHR23B (mouse homolog of the yeast rad23 gene); 4, p58/GTA (galactosyltransferase-associated protein kinase or cdc2-related protein kinase); 5, cyclin D3; 7, cyclin B1; 8, cyclin B2; and 9, cyclin A.

View larger version (52K): [in this window] [in a new window]   FIG. 6.   Regenerating transgenic livers exhibit premature and prolonged expression of genes involved in DNA replication and mitosis. Total RNA was prepared from regenerating wild-type and transgenic mouse livers, and RNase protection assays were used to analyze for expression of cyclin D1, p34cdc2 (cdc2), cyclin B1, C/EBP, and cyclophilin. Both cyclophilin RNase-protected bands were used as a normalization internal control (indicated by arrows), and representative RNase protection assays displaying the expression levels of these genes at various times after PH are shown. (A) Regenerating transgenic livers exhibit protracted expression of S phase-promoting cyclin D1 and premature expression of M phase promoting cdc2 and cyclin B1. In regenerating transgenic livers, protracted cyclin D1 expression occurs during hepatocyte DNA replication (32 and 36 h post-PH), and cyclin B1 expression is induced at 32 h after PH, which precedes detectable wild-type expression by 8 h. Maximal transgenic expression of cdc2 is observed at 32 h post-PH, which precedes induced wild-type expression by 8 h (see panel B). (B) Increased expression of cdc2 RNA in regenerating transgenic mouse liver. Shown is the mean induction of cdc2 expression in regenerating wild-type and transgenic livers with the standard deviation derived from three to six mice per time point (the asterisk indicates P < 0.05). (C) Regenerating transgenic livers exhibit protracted expression of S phase-promoting C/EBP transcription factor. RNase protection assay demonstrates protracted expression of the C/EBP mRNA during transgenic hepatocyte DNA replication which occurs 24 and 32 h post-PH.

A protracted increase in C/EBP expression is observed prior to transgenic hepatocyte DNA replication. Liver regeneration studies with C/EBP null mice demonstrate a 75% reduction in hepatocyte DNA replication and reduced expression of proliferation and metabolic homeostasis genes (21). Because C/EBP plays an important role in mediating hepatocyte replication after partial PH, we examined its expression pattern in regenerating wild-type and transgenic livers. Regenerating wild-type livers exhibit a biphasic expression pattern with a sharp increase at 24 h post-PH and a second increase during hepatocyte replication at 40 h after PH (Fig. 6C). A more protracted induction of C/EBP expression was observed prior to transgenic hepatocyte DNA replication (24 and 28 h post-PH), and elevated C/EBP mRNA levels were maintained throughout the period of hepatocyte replication (Fig. 6C). The C/EBP expression profile was similar to that observed with cyclin D1, suggesting that their protracted expression contributes to the early onset of hepatocyte DNA replication in regenerating transgenic liver.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Two-thirds PH induces differentiated liver cells to reenter the cell cycle and results in cellular proliferation to attain the original liver size while simultaneously maintaining expression of many hepatocyte-specific genes required for organ function (15, 38, 46). Although immediate-early expression of transcription factors is critical for initiating hepatocyte replication after PH, the transcriptional mechanisms involved in later stages of hepatocyte proliferation remain uncharacterized. HFH-11B is a candidate transcription factor involved in hepatocyte cell cycle progression during liver regeneration because its expression is reactivated as a delayed-early transcription factor (57). In this study, we used the TTR promoter to drive earlier hepatocyte expression of the HFH-11B transgene to test the hypothesis that premature hepatocyte expression of HFH-11B would alter the kinetics of hepatocyte proliferation after PH. Consistent with this hypothesis, when HFH-11B was expressed earlier in regenerating hepatocytes, both DNA replication and mitosis were accelerated by 8 h compared to regenerating wild-type hepatocytes.

Although HFH-11B is abundantly expressed in nonregenerating transgenic livers, we did not observe any aberrant hepatocyte replication in adult transgenic hepatocytes. Immunohistochemistry staining of nonregenerating liver reveals that the HFH-11B transgene protein was retained in the cytoplasm and that hepatocyte nuclear localization of HFH-11B requires mitogenic signalling induced during liver regeneration. The earlier expression of the HFH-11B transgene protein in regenerating transgenic liver, however, elicited an 8-h acceleration in its hepatocyte nuclear localization. These liver regeneration studies suggest that there is a limiting amount of HFH-11B protein prior to DNA replication and that its premature nuclear availability will accelerate hepatocyte proliferation. These results are thus consistent with the hypothesis that posttranslational modification of the HFH-11B protein is required for its nuclear localization, which ultimately allows for transcriptional activation of proliferation target genes. During liver regeneration, tyrosine phosphorylation of the stat3 protein directly or phosphorylation of the IB protein, causing its dissociation from NF-B, induces nuclear translocation of these transcription factors and subsequent activation of target genes (1, 7, 12). Previous studies have demonstrated that the HFH-11B C-terminal activation domain is phosphorylated by M-phase specific kinases, as evidenced by immunohistochemical detection with the MPM2 monoclonal antibody (52). Although the mechanism of HFH-11B nuclear localization during liver regeneration remains unknown, it is likely that protein phosphorylation regulates HFH-11B nuclear translocation.

The analysis of wild-type and transgenic regenerating liver RNA by differential hybridization of cDNA array blots and RNase protection assays established that the HFH-11B transgene stimulated expression of genes involved in cell cycle progression. Regenerating transgenic livers exhibited a more protracted expression of the S phase-promoting C/EBP, cyclin D1, and cyclin D3 genes, which preceded the onset of hepatocyte DNA replication (24 to 36 h post-PH). Although nuclear translocation of the HFH-11B transgene protein occurs by 24 h post-PH, which precedes induction of cyclin D1 expression (Fig. 2 and 6), the onset of cyclin D1 expression occurs at the same time as was observed with regenerating wild-type liver. Premature expression of HFH-11B did sustain expression of cyclin D1 through DNA replication (between 28 and 36 h post-PH), however, suggesting that it plays a role in the maintenance of cyclin D expression. Parallel to the accelerated entry of regenerating transgenic hepatocytes into mitosis, an 8-h-earlier induction of cyclin B1, cdc2, and B2 gene expression was observed during the period of transgenic hepatocyte DNA replication. In contrast to the maintenance of cyclin D1 expression, these results suggest that premature HFH-11B expression elicits earlier expression of mitosis-promoting genes (cyclin B and cdc2) in regenerating transgenic livers. Earlier or protracted expression of the cyclin B, cyclin D, cdc2, and C/EBP genes in regenerating TTR-HFH-11B transgenic livers represents a plausible mechanism for accelerating hepatocyte cell cycle progression because liver regeneration studies with IL-6, C/EBP, and crem knockout mouse models demonstrate that induction of these genes is essential for mediating hepatocyte proliferation (8, 21, 44). The TTR-HFH-11B liver regeneration phenotype is therefore most similar to that seen in albumin-c-myc transgenic mice, which display a 10-h acceleration in the onset of hepatocyte proliferation after PH, which is correlated with earlier expression of cyclin A and cdc2 genes (14). It is also interesting to note that at 24 h post-PH, we observe a sixfold stimulation of N-myc and c-myc gene expression compared with wild-type livers at similar times after PH (data not shown). This result suggests that nuclear translocation of the HFH-11B protein may be maintaining expression of the myc family members, which exhibit biphasic expression during liver regeneration (4, 35). Furthermore, our transgenic liver regeneration studies are consistent with the polyploid phenotype of hfh11/trident-deficient embryonic hepatocytes, suggesting that HFH-11/Trident plays a role in coordinating the timing of DNA replication and mitosis (29). Regulation of HFH-11B nuclear translocation may therefore play a pivotal role in mediating hepatocyte entry from the G₁ to S phase of the cell cycle, as well as inducing genes required for the initiation of mitosis. Furthermore, preliminary studies demonstrate that diminished postnatal hepatocyte proliferation coincides with the downregulation of HFH-11 expression (42a), which is consistent with its function in mediating hepatocyte proliferation during liver regeneration.

It is equally interesting to note that ectopic HFH-11B expression also causes changes in expression of several genes involved in the DNA repair pathway (XRCC1, mHR21spA, and mHR23B). There is strong evidence that DNA repair activity is increased in proliferating hepatocytes during liver regeneration. Radiation-induced DNA damage is more proficiently repaired in regenerating hepatocytes compared to quiescent liver (45), and elevated levels of homologous DNA recombination activity are observed with nuclear protein extracts prepared from regenerating rat liver (47). The premature expression of the DNA repair XRCC1, mHR21spA, and mHR23B genes in regenerating TTR-HFH-11B transgenic livers will therefore provide the proper environment for enhancement of hepatocyte replication by providing mechanisms to effectively repair DNA damage. Furthermore, HFH-11 displays abundant expression in thymus and testis (27, 56, 57), which correlates with high levels of expression of these DNA repair genes (36, 49, 58). More recent genetic data demonstrate that excision repair cross complementing-1 (ERCC-1)-deficient hepatocytes are prematurely polyploid, and ultimately these knockout mice die perinatally from liver failure (37). To further support the potential role of HFH-11/Trident in regulating DNA repair genes, Clever and colleagues have shown that hfh11/trident-deficient mice died perinatally with premature polyploid phenotypes in embryonic cardiomyocytes and hepatocytes (29). These results indicate that loss of HFH-11/Trident function causes the uncoupling of DNA synthesis from mitosis and suggest the hypothesis that HFH-11/Trident may regulate genes involved in cell cycle checkpoint control. The fact that premature expression of HFH-11/Trident in regenerating liver accelerates the timing of both hepatocyte DNA replication and mitosis further supports its role in cell cycle regulation.