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Protein tyrosine phosphatase of liver regeneration-1 (Prl-1) is an immediate-early gene that is significantly induced during liver regeneration. Several in vitro studies have suggested that Prl-1 is important for the regulation of cell cycle progression. To evaluate its function in liver regeneration, we ablated the Prl-1 gene specifically in mouse hepatocytes using the Cre-loxP system. Prl-1 mutant mice (Prl-1(loxP/loxP);AlfpCre) appeared normal and fertile. Liver size and metabolic function in Prl-1 mutants were comparable to controls, indicating that Prl-1 is dispensable for liver development, postnatal growth, and hepatocyte differentiation. Mutant mice demonstrated a delay in DNA synthesis after 70% partial hepatectomy, although ultimate liver mass restoration was not affected. At 40 h posthepatectomy, reduced protein levels of the cell cycle regulators cyclin E, cyclin A2, cyclin B1, and cyclin-dependent kinase 1 were observed in Prl-1 mutant liver. Investigation of the major signaling pathways involved in liver regeneration demonstrated that phosphorylation of protein kinase B (AKT) and signal transducer and activator of transcription (STAT) 3 were significantly reduced at 40 h posthepatectomy in Prl-1 mutants. Taken together, this study provides evidence that Prl-1 is required for proper timing of liver regeneration after partial hepatectomy. Prl-1 promotes G1/S progression via modulating expression of several cell cycle regulators through activation of the AKT and STAT3 signaling pathway., (Copyright © 2015 the American Physiological Society.)

Tyrosine phosphatase PRL-1 is one of the immediate-early genes up-regulated during liver regeneration and is apparently involved in cell proliferation. Previously, we have demonstrated that halofuginone, an inhibitor of collagen type I synthesis, prevents liver fibrosis and improves cirrhotic liver regeneration. In this study, we evaluated the effect of halofuginone on PRL-1 expression, its cellular localization in vitro and during liver regeneration, and fibrosis progression in vivo. In culture, halofuginone increased PRL-1 expression in primary rat hepatocytes and in hepatocellular carcinoma (HCC) cell lines, the former being more sensitive to halofuginone. The halofuginone-dependent increase in PRL-1 gene expression was correlated with an increase in the transcription factor early growth response-1 (Egr-1) and inversely correlated with the inhibition of cell proliferation. Halofuginone arrested HepG2 and Huh7 cell lines at the G1 phase, whereas Hep3B cells were arrested at G2/M, probably because of a reduction in the synthesis of cyclins D1 and B1 in all HCC cells and increased cyclin A in Hep3B cells. Halofuginone also affected the PRL-1 sub-cellular localization that was cell-cycle-dependent. In addition, halofuginone augmented PRL-1 expression in the remnant liver after partial hepatectomy and in chemically induced fibrosis in rats; this was accompanied by increased expression of insulin-like growth factor binding protein 1 (IGFBP-1), another immediate-early gene of regeneration. The regulation of the expression of the early genes of regeneration such as PRL-1 and IGFBP-1 is thus part of the mode of action of halofuginone and results in the prevention of liver fibrosis and improved cirrhotic liver regeneration.

Despite the growing incidence of obesity, knowledge of how this condition, as well as associated steatosis, affects liver regeneration remains scarce. Many previous studies have used models of steatohepatitis or obesity induced by genetic alterations. In contrast, our studies on liver regeneration have focused on the effects of obesity resulting solely from high amounts of fat in the diet. This model more closely reflects the detrimental effects of dietary habits responsible for increased morbidity due to obesity and its complications in well-developed Western societies. Impairment of liver regeneration was observed after partial hepatectomy in mice fed a high-fat diet. Fatty livers were more susceptible to posthepatectomy damage and failure. The underlying molecular mechanism was associated with increased inhibitor of nuclear factor-kappa B alpha (IkappaBalpha) expression, which inhibited nuclear factor-kappa B (NF-kappaB) activation and induction of its target genes, cyclin D1 and Bcl-xL, increasing sensitivity to apoptosis initiated by elevated tumor necrosis factor-alpha. In addition, since mice fed with a high-fat diet have higher leptin levels caused by increased adiposity, our work supports the hypothesis that the impairment of regeneration previously seen in genetically obese mice indeed results from liver steatosis rather than the disruption of leptin signaling. In conclusion, high fat in the diet impairs liver regeneration and predisposes steatotic livers to increased injury through IkappaBalpha overexpression and subsequent NF-kappaB inhibition.

The unusual regenerative properties of the liver are a logical adaptation by organisms, as the liver is the main detoxifying organ of the body and is likely to be injured by ingested toxins. The numerous cytokine- and growth-factor-mediated pathways that are involved in regulating liver regeneration are being successfully dissected using molecular and genetic approaches. So what is known about this process at present and which questions remain?

Interleukin-6 (IL-6) is required for normal liver regeneration, but the specific cellular source of this growth factor is unknown. We investigated whether this signal originates from the resident macrophage, the Kupffer cell. Using a murine model of bone marrow transplantation, we replaced recipient bone marrow-derived cells, including Kupffer cells, with cells of donor genetic phenotype. Recipients deficient in IL-6 (IL-6(-/-)) were lethally irradiated, then rescued with 10(7) donor bone marrow cells capable of expressing IL-6 (IL-6(+/+)). Conversely, IL-6(+/+) recipients received IL-6(-/-) marrow. Successful engraftment was measured by the presence of the Y chromosome SRY locus in the livers of female recipients receiving male marrow, in situ IL-6 expression by Kupffer cells, and up-regulation of IL-6 in splenocytes after activation with lipopolysaccharide (LPS). Kupffer cell isolation in IL-6(-/-) females receiving IL-6(+/+) male marrow clearly showed the presence of the SRY locus and IL-6 disrupted allele, whereas males receiving female marrow demonstrated no SRY or IL-6 signals, confirming the extent of replacement. Replacement of these cells in IL-6(-/-) mice with IL-6(+/+) bone marrow successfully restored the regenerative response after partial hepatectomy (PHx) as indicated by signal transduction and activator of transcription 3 (STAT3) activation and hepatocyte DNA replication. Alternatively, complete replacement of Kupffer cells in IL-6(+/+) mice by transplantation with IL-6(-/-) cells significantly inhibited liver regeneration and was partially restored by administration of IL-6. This investigation demonstrates a paracrine mechanism by which cells of bone marrow origin, most likely Kupffer cells, regulate the regenerative capacity of the hepatocyte through IL-6 expression.

Background: Every liver that is procured, stored, and transplanted experiences injury from cold ischemia and reperfusion. Most recover quickly, but some grafts sustain enough injury to result in prolonged organ dysfunction or require retransplantation. The molecular mechanisms involved in early graft function and recovery following cold ischemia and reperfusion (I/R) after liver transplantation have not been well defined. Interleukin (IL)-6 is a critical factor in the mitogenic response within the liver, and is important for cell cycle progression and protection from injury. Activation of the latent transcription factor, STAT3, is dependent on IL-6 release. The role of the IL-6/STAT3 pathway and hepatocellular regeneration in graft recovery and cell cycle progression following cold ischemia and reperfusion was studied in a rat liver transplant orthotopic (OLT) model. Methods. Rat OLT was performed in a syngeneic model. The presence, time course, and magnitude of expression of IL-6, STAT3 activation, and upregulation of target immediate early genes were determined in liver grafts with minimal (<1 h) and prolonged (12 h) cold preservation times followed by transplantation. Progression of the cell cycle and replication was confirmed by BrdU uptake., Results: Prolonged cold ischemia resulted in increased IL-6 expression and STAT3 activation. This correlated with upregulation of junB, c-fos, c-myc, and c-jun, immediate early genes associated with hepatic regeneration. Extensive DNA replication was present in livers with 12-h ischemia, demonstrating successful completion of the cell cycle., Conclusions: The participation of the IL-6/STAT3 pathway leading to cell cycle progression and regeneration is an important component in the recovery of organs immediately following cold preservation and transplantation., (Copyright 2001 Academic Press.)

Nuclear factor kappaB (NF-kappaB) is rapidly activated during liver regeneration following partial hepatectomy or carbon tetrachloride (CCl(4))-mediated liver injury and is felt to be important in the antiapoptotic and regenerative responses. After partial hepatectomy, livers of mice deficient in the p50 subunit of NF-kappaB (p50(-/-)) showed a loss of NF-kappaB and decreased STAT3 transcription factor DNA binding activities. However, nuclear levels of the NF-kappaB p65 subunit were increased and peaked earlier in p50(-/-) livers. Both messenger RNA and cytoplasmic protein levels of the NF-kappaB inhibitor IkappaBalpha were lower in p50(-/-) livers, potentially accounting for the increase in p65 protein. Small effects on gene expression posthepatectomy were observed in p50(-/-) livers, but no effects were seen on hepatocyte DNA synthetic or mitotic responses, serum enzyme levels, or overall liver mass restoration. After CCl(4) treatment, hepatocyte DNA synthesis and mitosis and serum enzyme levels were similar in p50(-/-) and p50(+/+) mice, and histologic analysis indicated a slight decrease in overall damage in p50(-/-) livers. After injection of Fas antibody, p50(-/-) livers showed an earlier onset of nuclear changes consistent with apoptosis. These data indicate that absence of p50 affects certain protein and gene activation pathways following partial hepatectomy, CCl(4), and Fas treatment but does not impair overall liver regeneration. Interleukin 6 (IL-6) levels were reduced but still adequate to support regeneration. We hypothesize that increased levels of the NF-kappaB p65 subunit in p50(-/-) livers may provide compensation for the absence of p50, thereby allowing normal liver regeneration and repair following liver injury.

The insulin-like growth factor binding protein-1 (IGFBP-1) gene is highly expressed in fetal, perinatal, and regenerating liver. Up-regulation is transcriptionally mediated in regenerating liver and occurs in the first few minutes to hours after partial hepatectomy. In transgenic mice a 970-bp region from -776 to +151 of the IGFBP-1 promoter was sufficient for tissue-specific and induced expression of the gene in fetal and hepatectomized livers. However weak and/or poorly regulated expression in some transgenic lines suggested the existence of other regulatory regions. Here, genomic clones containing large regions 5' of the mouse IGFBP-1 gene sequence were isolated, subcloned, and sequenced. Deoxyribonuclease I (DNaseI) hypersensitivity analyses identified clusters of tissue-specific nuclease-sensitive sites in the promoter region, -100 to -300, -2,300, -3,100, and -5,000 along with other weak sites. After partial hepatectomy, enhanced sensitivity and/or novel sites were detected in the -100/-300, -5,000, and -3,100 regions, the promoter region remaining the most hypersensitive. A subset of these sites was present in fetal and perinatal livers. Novel tissue-specific sites that interacted with C/EBP and hepatic nuclear factor 3 (HNF3) transcription factors were identified in the -3,100 region. A hepatectomy-induced DNA binding complex containing the transcription factor USF1 was identified within the -100 to -300 region of the promoter. These results suggested that a complex array of tissue-specific and hepatic proliferation-induced transcription factors combine to regulate both the proximal promoter and more distal regulatory elements of the IGFBP-1 gene.

Partial hepatectomy and toxic liver damage induce signals in the liver that result in rapid changes in the transcriptional milieu, including activation of latent transcription factors NF-kappa B and STAT3, and induction of expression of early growth response genes. Several of these changes within hepatocytes, including STAT3 and NF-kappa B induction are dependent on the cytokines, TNF alpha and interleukin-6 (IL-6), that are presumably released from non-parenchymal liver cells within minutes of the hepatectomy. IL-6 is a critical factor in the mitogenic response during liver regeneration and is important for both cell cycle progression and protection from liver injury. However, it is not a complete factor in that it is responsible for only a subset of the gene expression changes that occur after hepatectomy and is insufficient alone to cause hepatic DNA synthesis. C/EBP beta, a leucine zipper transcription factor, acts in an IL-6 independent fashion to induce a separate set of genes and proteins and is also required for normal liver regeneration. Moreover, some early growth response genes such as PRL-1, which encodes a nuclear protein tyrosine phosphatase, are induced normally in the absence of C/EBP beta and IL-6 and highlight the role of other transcriptional complexes such as Egr-1 in the early phases of liver regeneration. Thus, cytokine-dependent and -independent pathways act cooperatively to control the complex series of events that result in liver regeneration. The requirement for multiple signals also protects the liver from undergoing hyperplasia in the absence of a compensatory need.

Arg-Ser-rich domain-containing proteins (SR proteins), a family of splicing factors, can regulate pre-mRNA alternative splicing in a concentration dependent manner. Thus, the relative expression of various SR proteins may play an important role in alternative splicing regulation. HRS/SRp40, an SR protein and delayed early gene in liver regeneration, can mediate alternative splicing of fibronectin mRNA. Here we determined that transcription of the HRS/SRp40 gene is induced about 5-fold during liver regeneration, similar to the level of steady-state mRNA. We found that both mouse and human HRS promoters lack TATA and CAAT boxes. The mouse promoter region from -130 to -18, which contains highly conserved GA-binding protein (GABP) and YY1 binding sites, conferred high transcriptional activity. While GABPalpha/GABPbeta heterodimer transactivated the HRS promoter, YY1 functioned as a repressor. During liver regeneration, the relative amount of GABPalpha/GABPbeta heterodimer increased 3-fold, and YY1 changed little, which could partially account for the increase in HRS gene transcription. Interleukin-6, a critical mitogenic component of liver regeneration, was able to relieve the repressive activity of the YY1 site within the HRS promoter. The combined effect of small changes in the level of existing transcription factors and mitogenic signals may explain the transcriptional activation of the HRS gene during cell growth.

After two-thirds hepatectomy, normally quiescent liver cells are stimulated to reenter the cell cycle and proliferate to restore the original liver mass. The level of bZIP transcription factor CCAAT enhancer-binding protein beta (C/EBPbeta) increases in the liver during the period of cell proliferation. The significance of this change in C/EBP expression is not understood. To determine the role of C/EBPbeta in the regenerating liver, we examined the regenerative response after partial hepatectomy in mice that contain a targeted disruption of the C/EBPbeta gene. Posthepatectomy, hepatocyte DNA synthesis was decreased to 25% of normal in C/EBPbeta -/- mice. The reduced regenerative response was associated with a prolonged period of hypoglycemia that was independent of expression of C/EBPalpha protein and gluconeogenic genes. C/EBPbeta -/- livers showed reduced expression of immediate-early growth-control genes including the Egr-1 transcription factor, mitogen-activated protein kinase protein tyrosine phosphatase (MKP-1), and HRS, a delayed-early gene that encodes an mRNA splicing protein. Cyclin B and E gene expression were dramatically reduced in C/EBPbeta -/- livers whereas cyclin D1 expression was normal. The abnormalities in immediate-early gene expression in C/EBPbeta -/- livers were distinct from those seen in IL-6 -/- livers. These data link C/EBPbeta to the activation of metabolic and growth response pathways in the regenerating liver and demonstrate that C/EBPbeta is required for a normal proliferative response.

Serine-arginine (SR)-rich proteins are believed to be important in mediating alternative pre-mRNA splicing. HRS/SRp40 expression is elevated in liver cell proliferation during development, regeneration, and oncogenesis. We tested whether HRS expression correlates with the appearance of alternatively spliced fibronectin transcripts during liver growth. HRS was highly expressed during the proliferative phase of liver development, correlating with expression of the fibronectin EIIIB alternative exon. In regenerating liver, HRS protein was induced in a time course consistent with the observed increase in fibronectin transcripts containing the EIIIB exon, particularly in nonparenchymal liver cells. Furthermore, in an in vivo assay, HRS, and not other SR proteins, directly mediated EIIIB exon inclusion in the fibronectin transcript. This alternative splicing was dependent on a purine-rich region within the EIIIB exon to which HRS specifically bound. We have established that HRS has the potential to contribute to the regulation of fibronectin pre-mRNA splicing during liver growth. Changes in fibronectin forms may be important in modifying liver architecture during the proliferative response, thus providing a potential mechanism by which SR proteins may participate in cellular growth control.

Liver regeneration stimulated by a loss of liver mass leads to hepatocyte and nonparenchymal cell proliferation and rapid restoration of liver parenchyma. Mice with targeted disruption of the interleukin-6 (IL-6) gene had impaired liver regeneration characterized by liver necrosis and failure. There was a blunted DNA synthetic response in hepatocytes of these mice but not in nonparenchymal liver cells. Furthermore, there were discrete G1 phase (prereplicative stage in the cell cycle) abnormalities including absence of STAT3 (signal transducer and activator of transcription protein 3) activation and depressed AP-1, Myc, and cyclin D1 expression. Treatment of IL-6-deficient mice with a single preoperative dose of IL-6 returned STAT3 binding, gene expression, and hepatocyte proliferation to near normal and prevented liver damage, establishing that IL-6 is a critical component of the regenerative response.

Liver regeneration following partial hepatectomy results in proliferation of virtually all of the cells in the remnant liver, leading to restitution of mass within a few days. The regenerating liver maintains its functional capacity, and liver-specific factors, such as C/EBP's, continue to be expressed. Growth-induced transcription factors Stat3 and NF-KB regulate the expression of growth-response genes, ultimately leading to cell growth. Although well-modulated levels of cytokines may promote liver regeneration posthepatectomy, aberrant production of cytokines that can be seen in hepatitis, cirrhosis, and after liver transplantation may lead to hepatomas, fibrosis, and liver failure.

Determining what factors are responsible for initiating regeneration following partial hepatectomy or toxic damage, and how the liver maintains differentiated functions while the hepatocytes are undergoing cellular proliferation are central issues in understanding the molecular bases of liver regeneration. Examination of the transcriptional milieu in the regenerating liver provides clues to the answers to these questions. Growth factor-generated intracellular signals that trigger liver regeneration result in activation via posttranslational modifications of latent, normally inactive transcription factors that preexist in the liver. Two transcription factors that are activated by this mechanism include posthepatectomy factor/nuclear factor-kappa B) and Stat3. Because cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-l (IL-1), and IL-6 can induce these factors in the liver, the finding of activated Stat3 and PHF/NF-kappa B suggests that these cytokines may play a role in some aspects of growth regulation during liver regeneration. Rapidly induced transcription factors, Stat3, PHF/NF-kappa B, and others are responsible for activation of the primary growth response or immediate-early genes, which play a role in regulating later phases of cell growth in regenerating liver and other mitogen-activated cells. Immediate-early genes encode many members of diverse transcription factor families including the Jun-Fos-LRF-1, nuclear receptor, and myc families to name a few. In this way a transcriptional cascade is established during the G1 phase of liver regeneration. Coexisting with these induced factors are liver-specific transcription factors such as the CAAT enhancer binding proteins and hepatocyte nuclear factors, which may interact with growth-induced factors to help the liver maintain metabolic homeostasis during regeneration. As a result the liver is able to accomplish the goals of reestablishing its mass while it maintains its functional capacity during regeneration.

During the period of rapid cell growth which follows a two-thirds partial hepatectomy, the liver is able to compensate for the acute loss of two-thirds of its mass to maintain serum glucose levels and many of its differentiation-specific functions. However certain hepatic transcription factors, C/EBP alpha and beta, which are important for establishment and maintenance of the differentiated state, have been shown to be antagonistic to cellular proliferation. To study the interplay between differentiation and cell growth in the liver regeneration model of hepatocyte proliferation, we characterized the expression of C/EBP alpha and beta transcription factors throughout the temporal course of liver regeneration. As determined by immunoblot, the level of C/EBP alpha decreases more than twofold during the mid to late G1 and S phase (8-24 h after hepatectomy) coordinately with a threefold increase in expression of C/EBP beta. Renormalization of the levels of these proteins occurs after the major proliferative phase. This inverse regulation of C/EBP alpha and beta results in up to a sevenfold increase in the beta / alpha DNA binding ratio between 3 and 24 h after hepatectomy that may have an important impact on target gene regulation. However, total C/EBP binding activity in nuclear extracts remains relatively constant during the 7-d period after hepatectomy. By immunohistochemistry, both C/EBP alpha and beta are expressed in virtually all hepatocyte nuclei throughout the liver during the temporal course of liver regeneration, and there is no exclusion of expression from hepatocytes that are expressing immediate-early gene products or undergoing DNA synthesis. The persistent expression of C/EBP alpha and beta isoforms predicts that C/EBP proteins contribute to the function of hepatocytes during physiologic growth and that significant amounts of these proteins do not inhibit progression of hepatocytes into S phase of the cell cycle.

The liver shows maximal cellular growth during fetal development and after partial hepatectomy. Exploring overlaps in gene expression patterns in these two types of hepatic growth may provide insight into common regulatory pathways. The expression of a large number of growth-induced and liver-specific genes induced in liver regeneration has been examined in the perinatal liver from several days prenatal to 4 weeks postnatal when the major growth phase of the liver ceases. As in liver regeneration, many growth-induced genes, such as PRL-1 and beta-actin, are expressed at a high level throughout the temporal course of liver development and correlate with the proliferative state. The level of fetal liver expression of these genes is similar to peak expression found in the regenerating liver, suggesting that common pathways of transcriptional regulation exist in the two types of proliferation. A subset of liver-restricted immediate-early genes including, IGFBP-1, CL-6, and glucose-6-phosphatase (G6Pase) are induced in regenerating liver and may be important in maintaining hepatic metabolism during regeneration. In developing liver, these genes are expressed primarily in the perinatal period but, unlike the regenerating liver, are not coinduced. For instance, at birth, G6Pase is induced, whereas CL-6 is downregulated. In situ analyses confirm that a proliferation associated gene PRL-1 is expressed in multiple cell types throughout the developing liver, whereas the expression of liver-specific genes is confined to hepatocytes. Taken together, these findings imply that significant similarities and differences in transcriptional regulation and hormonal milieu exist in liver during regeneration and development.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver regeneration in response to partial hepatectomy is a physiological growth response observed in the intact animal. Understanding the early signals that trigger liver regeneration is of vital importance to understand the liver's response to injury. It has been observed that several growth factors and cytokines, including epidermal growth factor (EGF) and interleukin-6 (IL-6), can activate members of the signal transducers and activators of transcription (Stat) family of transcription factors resulting in tyrosine phosphorylation of these factors, nuclear translocation, and an active DNA binding transcriptional complex. Because Stat3 participates in the regulation of primary growth response genes, we wondered if it is induced in the early phase of liver regeneration. We found that Stat3 DNA-binding activity is increased in the remnant liver within 30 minutes of partial hepatectomy and peaks at more than 30-fold at 3 hours. This induction is not observed after sham surgery. The induction of Stat3 appears to be part of the initial response of the remnant liver to partial hepatectomy, because it occurs in the presence of cycloheximide-mediated protein synthesis blockade. Activation of Stat3 is unusual, because it extends beyond the immediate-early time period and remains near peak level at 5 hours posthepatectomy. Although insulin-treated H35 cells activate many of the same immediate-early genes as regenerating liver, Stat3 is not induced in these cells. Because Stat factors are known to be inactivated by protein tyrosine phosphatases (PTPase), we showed that a PTPase is able to eliminate the DNA binding of hepatic Stat3.(ABSTRACT TRUNCATED AT 250 WORDS)

The regenerating liver after partial hepatectomy is one of the few physiologic models of cellular proliferation in the adult animal. During hepatic regeneration, the animal is able to maintain metabolic homeostasis despite the acute loss of two thirds of hepatic tissue. In examining the molecular mechanisms regulating hepatic regeneration, we isolated novel immediate-early genes that are rapidly induced as the remnant liver undergoes the transition from its normal quiescent state into the G1 phase of the cell cycle. One of the most rapidly and highly induced genes which we initially termed RL-1, encodes rat glucose-6-phosphatase (rG6Pase). G6Pase mRNA peaks at 30 min and 36-48 h after hepatectomy correlating with the first and second rounds of cell division. This finding is compatible with studies that showed that G6Pase enzyme activity increases during liver regeneration. However, the increase in G6Pase mRNA is much more dramatic, indicating that it is a more sensitive indicator of this regulation. G6Pase gene expression peaks in the perinatal time period in the liver and remains elevated during the first month of life. The expression of the G6Pase gene is also dramatically elevated in BB diabetic rats, again higher than the enzyme elevation, and its relative induction after partial hepatectomy is blunted in these animals. Insulin treatment of partially hepatectomized diabetic animals downregulates the expression of G6Pase mRNA. Using specific antibodies against G6Pase, we detect a 36-kD G6Pase protein, and its level is elevated in regenerating and diabetic livers. The pattern of G6Pase mRNA expression appears to reflect similar changes in insulin and glucagon levels which accompany diabetes and hepatic proliferation. The elevation of G6Pase expression in these conditions is indicative of its importance as a regulator of glucose homeostasis in normal and abnormal physiologic states.

The liver represents one of the few organs in the intact animal that has the capacity to regenerate following injury or partial hepatectomy. One of the earliest responses that has been detected in the remnant liver is the activation of post-hepatectomy factor(s) (PHF), a kappa B site DNA binding activity. We reasoned that understanding the molecular nature of PHF might provide insight into what triggers liver regeneration. We found that PHF is rapidly activated and turned over in the regenerating liver, demonstrating peak activity at 30 min post-hepatectomy and virtual disappearance by 1 h. As determined by supershift, cross-linking, and cross-linking/immunoprecipitation analyses, PHF contains intact p50/p65nuclear factor kappa B (NF-kappa B) subunits. To explore the basis for activation of PHF/NF-kappa B in the regenerating liver, we determined the level of individual Rel family subunits in the nuclei of normal and regenerating liver cells. We found evidence for nuclear translocation of p65/RelA, but other Rel family proteins including p50/NF-kappa B1 and p52/NF-kappa B2 are present at a low level in the nuclei of cells at a constitutive level pre- and post-hepatectomy and appear not to form DNA binding homodimers. The level of I kappa B-alpha falls slightly then increases at 3 h post-hepatectomy in concert with the induction of its mRNA. As demonstrated by the induction of I kappa B-alpha mRNA in hepatocytes in situ and identification of PHF/NF-kappa B in cultured hepatocytes, PHF/NF-kappa B is localized primarily in hepatocytes in the regenerating liver. This represents one of the few examples of NF-kappa B activation in the intact animal in a non-hematopoietic cell type. The activation of PHF/NF-kappa B suggests a mechanism by which hepatocytes regulate their mitogenic program during liver regeneration.

Regenerating liver is a physiologic animal system in which p50/p65 nuclear factor kappa B (NF-kappa B) DNA binding activity is induced and rapidly disappears in the remnant liver within minutes of partial hepatectomy. The activation of NF-kappa B may contribute to the mechanism by which hepatocytes regulate their mitogenic program during liver regeneration. A p50/NF-kappa B1-p35/RelA heterodimer that contains a proteolyzed amino-terminal DNA binding fragment of p65/RelA is also present in the nuclei of hepatic cells within minutes of partial hepatectomy. In the absence of I kappa B-alpha resynthesis, turnover of NF-kappa B occurs via conversion of p50/NF-kappa B1-p65/RelA to a p50/NF-kappa B1-p35/RelA DNA binding complex. Proteolytic conversion of p65/RelA into p35/RelA and subsequent degradation of p35/RelA account at least in part for the rapid turnover of NF-kappa B in the cell nuclei. Nuclear proteolysis provides a potential mechanism for tightly regulating the level of active NF-kappa B.

Liver regeneration factor belongs to the leucine-zipper family of transcription factors. It was originally cloned and characterized through differential screening of a regenerating rat liver cDNA library. The mRNA for liver regeneration factor-1 is barely detectable in normal rat liver but is dramatically induced after two-thirds hepatectomy, with a peak 1 to 3 hr after surgery. The nature of the signaling molecule(s) for this rapid induction is not known. It has been suggested that the liver regeneration factor-1 protein product, through complex interactions with other transcription factors such as c-Jun and Jun-B, controls expression of genes that are required during the G1 phase of hepatic growth. Hepatocyte growth factor has been shown to be the most potent mitogen for hepatocytes in vitro and in vivo. Plasma levels of hepatocyte growth factor rapidly (within 30 min) increase after loss of hepatic parenchyma induced by partial hepatectomy or carbon tetrachloride treatment. It has been postulated that hepatocyte growth factor plays a crucial role in stimulating the hepatocyte to enter the cell cycle. In this communication, we report that addition of pure hepatocyte growth factor to primary cultures of rat hepatocytes in the absence of serum and insulin results in rapid and transient induction of liver regeneration factor-1 mRNA (more than 20-fold) with a peak of expression 1 hr after treatment. The levels of jun-B and c-fos mRNAs, which are also known to be induced during the early hours of liver regeneration, were also increased after treatment of isolated hepatocytes with hepatocyte growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver regeneration following partial hepatectomy provides one of the few systems for analysis of mitogenesis in the fully developed, intact animal. Immediate-early growth response genes, induced in the absence of prior protein synthesis, play an important regulatory role in the regenerative process. During screening of a subtracted cDNA library of immediate-early genes induced during liver regeneration, a novel member of the thyroid/steroid receptor superfamily, RNR-1 (regenerating liver nuclear receptor), was identified. This gene is not expressed in quiescent liver but is rapidly induced following partial hepatectomy and is specific to hepatic growth as it is not induced in other mitogen-treated cells. RNR-1 is also expressed in brain. A full-length cDNA clone of RNR-1 encodes a 66-kDa, 597-amino acid protein as verified by in vitro translation in reticulocyte lysate. RNR-1 is highly homologous to r-NGFI-B/m-Nur77 particularly in the DNA binding (94%) and putative ligand binding (59%) domains. Using a mobility shift assay, we have shown that RNR-1 specifically binds to the NGFI-B DNA half-site and forms a complex very similar in size to the Nur77 complex, suggesting that RNR-1 also may bind as a monomer. Consistent with this finding, the A box region important in mediating half-site binding is 100% conserved between r-NGFI-B/m-Nur77. Both RNR-1 and Nur77 strongly transactivate a reporter driven by a consensus r-NGFI-B/Nur77 binding site, and their effect together is additive. As both the RNR-1 and r-NGFI/m-nur77 genes are induced during liver regeneration, it is very possible that RNR-1 acts concomitantly with r-NGFI/m-Nur77 in regulating the expression of delayed-early genes during liver regeneration.

Liver regeneration is an important process that allows for recovery from hepatic injuries caused by viruses, toxins, ischemia, surgery, and transplantation. Previously, we identified > 70 immediate-early genes induced in regenerating liver after hepatectomy, 41 of which were novel. While it is expected that the proteins encoded by these genes may have important roles in regulating progression through the G1 phase of the cell cycle during regeneration, we were surprised to note that many of these "early" genes are expressed for extended periods during the hepatic growth response. Here we define several patterns of expression of immediate-early, delayed-early, and liver-specific genes during the 9-d period after hepatectomy. One pattern of induction parallels the major growth period of the liver that ends at 60-72 h after hepatectomy. A second pattern has two peaks coincident with the first and second G1 phases of the two hepatic cell cycles. A third group, which includes liver-specific genes such as C/EBP alpha, shows maximal expression after the growth period. Although the peak in DNA synthesis in nonparenchymal cells occur 24 h later than in hepatocytes, most of the genes studied demonstrate similar induction in both cell types. This finding suggests that the G0/G1 transition occurs simultaneously in all cells in the liver, but that the G1 phase of nonparenchymal cells may be relatively prolonged. Finally, we examined the expression of > 70 genes in clinical settings that could induce liver regeneration, including after perfusion in a donor liver, hepatic ischemia, and fulminant hepatic failure. We found that a small number of early and liver-specific genes were selectively activated in human livers under these conditions, and we thereby provide a potential means of measuring the caliber of the regenerative response in clinical situations.

In regenerating liver, a physiologically normal model of cell growth, LRF-1, JunB, c-Jun, and c-Fos among Jun/Fos/LRF-1 family members are induced posthepatectomy. In liver cells, high levels of c-Fos/c-Jun, c-Fos/JunB, LRF-1/c-Jun, and LRF-1/JunB complexes are present for several hours after the G0/G1 transition, and the relative level of LRF-1/JunB complexes increases during G1. We provide evidence for dramatic differences in promoter-specific activation by LRF-1- and c-Fos-containing complexes. LRF-1 in combination with either Jun protein strongly activates a cyclic AMP response element-containing promoter which c-Fos/Jun does not activate. LRF-1/c-Jun, c-Fos/c-Jun, and c-Fos/JunB activate specific AP-1 and ATF site-containing promoters, and in contrast, LRF-1/JunB potently represses c-Fos- and c-Jun-mediated activation of these promoters. Repression is dependent on a region in LRF-1 that includes amino acids 40 to 84 (domain R) and the basic/leucine zipper domain. As the relative level of LRF-1/JunB complexes increases posthepatectomy, c-Fos/Jun-mediated ATF and AP-1 site activation is likely to decrease with simultaneous transcriptional activation of the many liver-specific genes whose promoters contain cyclic AMP response element sites. Thus, through complex interactions among LRF-1, JunB, c-Jun, and c-Fos, control of delayed gene expression may be established for extended times during the G1 phase of hepatic growth.

The liver is one of the few adult tissues that has the capacity to regenerate following hepatectomy or toxic damage. In examining the early growth response during hepatic regeneration, we found that a highly induced immediate-early gene in regenerating liver encodes RL/IF-1 (regenerating liver inhibitory factor) and is the rat homolog of human MAD-3 and probably of chicken pp40. RL/IF-1 has I kappa B activity of broad specificity in that it inhibits the binding of p50-p65 NF-kappa B, c-Rel-p50, and RelB-p50, but not p50 homodimeric NF-kappa B, to kappa B sites. Like RL/IF-1, several members of the NF-kappa B and rel family of transcription factors are immediate-early genes in regenerating liver and mitogen-treated cells. We examined changes in kappa B site binding activity during liver regeneration and discovered a rapidly induced novel kappa B site-binding complex designated PHF [posthepatectomy factor(s)]. PHF is induced over 1,000-fold within minutes posthepatectomy in a protein synthesis-independent manner, with peak activity at 30 min, and is not induced by sham operation. PHF is distinct from p50-p65 NF-kappa B, which is present only in the inactive form in liver posthepatectomy. Although early PHF complexes do not interact strongly with anti-p50 antibodies, PHF complexes present later (3 to 5 h) posthepatectomy react strongly, suggesting that they contain a p50 NF-kappa B subunit. Unlike p50-p65 NF-kappa B, c-Rel-p50, and RelB-p50 complexes, PHF binding to kappa B sites is not inhibited by RL/IF-1. One role of RL/IF-1 in liver regeneration may be to inhibit p50-p65 NF-kappa B activity present in hepatic cells, allowing for the preferential binding of PHF to kappa B sites. Because PHF is induced immediately posthepatectomy in the absence of de novo protein synthesis, PHF could have a role in the regulation of liver-specific immediate-early genes in regenerating liver.

Liver regeneration provides one of the few systems for analysis of mitogenesis in the fully developed, intact animal. Several proteins have been identified as part of the primary growth response in regenerating liver and in mitogen-stimulated cells. Some of these proteins, such as the Jun and Fos families of transcription factors, are thought to have a role in activating transcription of genes expressed subsequently in the growth response. Through differential screening of a regenerating-liver cDNA library, we have identified a rapidly and highly induced gene encoding a 21-kDa leucine-zipper-containing protein that we have designated liver regeneration factor 1 (LRF-1). LRF-1 has no homology with other leucine-zipper proteins outside the basic and leucine-zipper domains. LRF-1 alone can bind DNA, but it preferentially forms heteromeric complexes with c-Jun and Jun-B and does not interact with c-Fos. In solution, it binds with highest affinity to cAMP response elements but also has affinity for related sites. In cotransfection studies, LRF-1 in combination with c-Jun strongly activates a c-Jun-responsive promoter. The induction of the LRF-1 gene in regenerating liver greatly increases the potential variety of heterodimeric combinations of leucine-zipper transcription factors. While LRF-1 mRNA is rapidly induced in the absence of protein synthesis, its peak induction is later than c-fos mRNA, suggesting that LRF-1 may regulate responsive genes at a later point in the cell cycle. As such, LRF-1 may have a unique and critical role in growth regulation of regenerating liver and mitogen-stimulated cells.

The liver is an epithelioid organ that can regenerate following partial hepatectomy. Although it is composed mainly of hepatocytes, it has a complex, multicellular architecture, implying that intercellular communications must exist during regeneration. As in other mitogen-stimulated cells, immediate-early growth response genes induced in the absence of prior protein synthesis are likely to play an important regulatory role in the regenerative process. Through differential screening of regenerating liver cDNA libraries, we found that one of the most highly expressed immediate-early genes in liver regeneration encodes the rat homolog of the low-molecular-weight insulinlike growth factor (IGF)-binding protein (IGFBP-1). This protein has been implicated in enhancing the mitogenic effect of IGF on tissues. IGFBP-1 gene induction is transcriptionally mediated and specific to regenerating liver, as the gene is not expressed in mitogen-stimulated fibroblasts. IGFBP-1 expression has been shown to increase under low-insulin conditions such as diabetes, and the complex regulation of expression is indicated by our finding that insulin treatment of H35 rat hepatoma cells, which induces proliferation, also causes a rapid decrease in transcription and expression of the IGFBP-1 gene. Of note, IGFBP-1 mRNA is abundant in fetal rat liver, implying that it participates in normal liver growth and development. Although regenerating liver cells continue to produce IGF-I, we did not detect IGF-I receptor mRNA during the first 24 h after hepatectomy. However, some IGFBPs may act to enhance the activity of IGF-I independently of IGF-I receptors. Thus, IGF-1 and IGFBPs may interact with hepatocytes or nonparenchymal liver cells, through either IGF-I or novel receptors. In this way, IGFBP-I and IGF-I could act in a paracrine and/or autocrine fashion in maintaining normal liver architecture during regeneration.

Liver regeneration provides a unique system for analysis of mitogenesis in intact, fully developed animals. Cellular immediate-early genes likely play an important role in cell cycle regulation and have been extensively studied in mitogen-stimulated fibroblasts lymphocytes but not in liver. We have begun to characterize the immediate-early growth response genes of mitogen-stimulated liver cells, specifically, regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells, and to address differences in growth response between different cell types. Through subtraction and differential screening of cDNA libraries from regenerating liver and insulin-treated H-35 cells, we have extensively characterized 341 differentially expressed clones and identified 52 immediate-early genes. These genes have been partially sequenced and subjected to Northern (RNA) blot analysis, and 41 appear to be novel. Surprisingly, two-thirds of these genes are also expressed in BALB/c 3T3 cells, but only 10 were identified in previous studies of 3T3 cells, and of these, 6 include well-known genes like jun and fos, and only 4 are novel. Approximately one-third of the immediate-early genes identified in mitogen-stimulated liver cells or serum-stimulated NIH 3T3 cells are expressed in a tissue-specific fashion, indicating that cell type-specific regulation of the proliferative response occurs during the immediate-early period. Our findings indicate that the immediate-early response is unusually complex for the first step in a regulatory cascade, suggesting that multiple pathways must be activated. The abundance of immediate-early genes and the highly varied pattern of their expression in different cell types suggest that the tissue specificity of the proliferative response arises from the particular set of these genes expressed in a given tissue.

Immediate-early genes, whose expression increases independent of de novo protein synthesis during the transition from quiescence to proliferation, are postulated to play important regulatory roles in the growth response. The complement of immediate-early genes expressed must depend on the milieu of preexisting transcription factors in the quiescent cell as well as the type of mitogenic stimulation and, thus, may differ between cell types. We have begun characterizing the immediate-early response in regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells in comparison with previously published results from mitogen-stimulated Balb/c 3T3 fibroblasts. The proliferating H-35 and regenerating liver cells maintain their similarity to quiescent liver as demonstrated by their continued production of the liver-specific albumin, CCAAT/enhancer binding protein, and phosphoenolpyruvate carboxykinase messenger RNAs (mRNA). Surprisingly, the phosphoenolpyruvate carboxykinase gene, which undergoes down-regulation in insulin-treated H-35 cells, was cloned by differential screening of a subtraction-enriched regenerating liver cDNA library and is an immediate-early gene in regenerating liver. H-35 cells treated with either insulin or phorbol 12-myristate 13-acetate express elevated levels of the jun genes, and phorbol 12-myristate 13-acetate pretreatment fails to abolish the insulin response, indicating that it does not depend on protein kinase C. jun family gene expression in regenerating liver differs from that in mitogen-treated fibroblasts in that the time course of expression of c-jun and junB is prolonged, and junD mRNA levels distinctly increase. Additionally, although c-fos and egr-1 mRNAs are expressed at elevated levels in stimulated liver cells, fos-B, fra-1, and egr-2 are not, which suggests that factors in addition to the serum response factor participate in the regulation of immediate-early gene induction. Interestingly, gene 33, which was cloned from a regenerating liver cDNA library by differential screening and lacks a recognizable serum response element, functions as an immediate-early gene in regenerating liver and in mitogen-treated H-35 and Balb/c 3T3 cells. These results suggest that gene 33 participates in the transition from quiescence to proliferation in many mitogen-treated cells in addition to its previously reported involvement in hormone responses. Overall, the results presented here suggest that the immediate-early response varies considerably between regenerating liver and mitogen-stimulated fibroblasts and could involve multiple, preexisting, tissue-specific, transcription-activating proteins.