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MGL-3196 is an oral, liver-targeted selective agonist for the thyroid hormone receptor-β (THR-β) that is being developed for the treatment of dyslipidemia. The safety profile and tolerability of THR-β agonist MGL-3196 was assessed in first-in humans studies, including a single ascending dose study (NCT01367873) in which MGL-3196 appeared safe at all doses tested. A two-week multiple dose study was conducted at doses of 5, 20, 50, 80, 100, and 200 mg per day in healthy subjects with mildly elevated low density lipoprotein (LDL) cholesterol (>110 mg/dL) (NCT01519531). MGL-3196 was well-tolerated at all doses with no dose-related adverse events or liver enzyme, ECG or vital-sign changes. At the highest dose, there was a reversible reduction of ∼20% in the level of pro-hormone, free thyroxine (free T4) that was significantly different from placebo (p < 0.0001) that may be explained by increased hepatic metabolism of T4. There was no change in thyrotropin (TSH) or triiodothyronine (free T3) or other evidence of central thyroid axis dysfunction at any dose. Doses ranging from 50 to 200 mg demonstrated highly statistically significant reductions relative to placebo of up to: 30% for LDL cholesterol (range, p = 0.05-<0.0001); 28% for non- high density lipoprotein (HDL) cholesterol (range, p = 0.027-0.0001); 24% for Apolipoprotein B (range, p = 0.008-0.0004), and statistical trends of up to 60% reduction in triglycerides (TG) (range, p = 0.13-0.016). The near maximal lipid effects were observed at a dose of 80 mg daily. In summary, in a two-week study in healthy volunteers with mild LDL cholesterol elevation, MGL-3196 appeared safe, was well-tolerated and showed a beneficial effect on lipid parameters., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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?

Stat3 is a vital transcription factor that is activated downstream of the gp130 receptor, primarily via IL-6 signaling in adult liver. A new study demonstrates that Stat3 provides hepatoprotection against Fas-mediated apoptotic liver damage by two mechanisms: direct inactivation of caspases and reduction of reactive oxygen species.

Acute liver failure caused by viral hepatitis or toxic damage involves both apoptotic and necrotic pathways. IGF binding protein-1 (IGFBP-1), a hepatocyte-derived secreted protein, is required for normal liver regeneration. To determine whether IGFBP-1 could prevent liver injury that entails direct stimulation of hepatocyte apoptosis, IGFBP-1(-/-) mice, IGFBP-1(+/+) mice, and mice pretreated with Ab's against IGFBP-1 were treated with a normally sublethal dose of Fas agonist. IGFBP-1 deficiency was associated with massive hepatocyte apoptosis and caspase activation within 3 hours of Fas agonist treatment, which could be corrected by pretreatment with IGFBP-1. IGFBP-1-deficient livers had enhanced signaling via the integrin receptor at early times (0.5 to 1 hour) after Fas agonist treatment accompanied by elevated activated matrix metalloproteinase-9 (MMP-9), a known target of fibronectin signaling and activator of TGF-beta. Within 3 hours of Fas agonist treatment, elevated expression of active TGF-beta1, a hepatocyte apoptogen, was observed in IGFBP-1-deficient livers that correlated with the appearance of the apoptotic process. Both MMP-9 and TGF-beta1 expression were suppressed by IGFBP-1 treatment, supporting their role in the apoptotic process. IGFBP-1(-/-) mice also displayed increased injury in a toxic hepatic injury model caused by CCl(4). These findings indicate that IGFBP-1 functions as a critical hepatic survival factor in the liver by reducing the level of proapoptotic signals.

Transforming growth factor-beta (TGF-beta) functions as an antiproliferative factor for hepatocytes. However, for unexplained reasons, hepatocytes become resistant to TGF-beta signals and can proliferate despite the presence of TGF-beta during liver regeneration. TGF-beta is up-regulated during liver regeneration, although it is not known whether it is active or latent. TGF-beta activity may be examined by assessing Smad activation, a downstream signaling pathway. Smad pathway activation during liver regeneration induced by partial hepatectomy or CC4 injury was examined by assessing the levels of phospho-Smad2 and Smad2-Smad4 complexes. We found that Smad proteins were slightly activated in quiescent liver, but that their activation was further enhanced in regenerating liver. Interestingly, TGF-beta/Smad pathway inhibitors (SnoN and Ski) were up-regulated during regeneration, and notably, SnoN was induced mainly in hepatocytes. SnoN and Ski are transcriptional repressors that may render some cells resistant to TGF-beta via binding Smad proteins. Complexes between SnoN, Ski, and the activated Smad proteins were detected from 2 to 120 h during the major proliferative phase in regenerating liver. Inhibitory complexes decreased after liver mass restitution (5-15 days), suggesting that persistently activated Smad proteins might participate in returning the liver to a quiescent state. Our data show that active TGF-beta/Smad signals are present during regeneration and suggest that SnoN/Ski induction might explain hepatocyte resistance to TGF-beta during the proliferative phase.

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. G(1) phase cyclins including cyclins D1 and E had lower expression levels in Alb(+) STAT3(fl/fl) livers, indicating an abnormal G(1) 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 vivo evidence that STAT3 promotes cell cycle progression and cell proliferation under physiological growth conditions.

Previous studies showed that following acute carbon tetrachloride (CCl(4)) treatment, interleukin-6 null (IL-6-/-) mice develop increased hepatocellular injury, defective regeneration, delayed wound healing, and increased hepatocyte apoptosis. Pretreatment with IL-6 prior to CCl(4) reduces injury, hepatocyte apoptosis, and accelerates regeneration in both IL-6-/- and +/+ livers. To demonstrate whether IL-6 can prevent liver injury that involves direct stimulation of hepatocyte apoptosis, IL-6-/- and +/+ mice were treated with the Fas agonist, Jo-2 mAb. At low Fas agonist doses, IL-6+/+ mice developed mild hepatic injury and survived, whereas IL-6-/- mice developed severe apoptotic hepatitis within 12 h and died. Pretreatment with IL-6 improved survival in IL-6-/- mice and reduced injury in both IL-6-/- and +/+ livers. The direct anti-apoptotic effects of IL-6 were demonstrated in vitro as IL-6 decreased Fas-mediated apoptosis in both IL-6-/- and +/+ primary hepatocyte cultures, and suggested that IL-6-/- hepatocytes have a pre-existing defect in anti-apoptotic pathways. After Fas activation, IL-6-/- livers demonstrated evidence of both proximal and distal alterations in the apoptotic pathways including elevated caspase 8 and 3 activation-associated fragments, and loss of cytochrome c staining. IL-6-/- livers had reduced pre-existing protein expression of the anti-apoptotic factors Bcl-2 and Bcl-xL as well as more rapid degradation of FLIP following Fas treatment that appeared to be post-transcriptionally regulated. FLIP is a crucial proximal inhibitor of caspase 8 activation in Fas, tumor necrosis factor, and DR3/DR4-mediated apoptosis, and Bcl-2 and Bcl-xL more downstream anti-apoptotic regulators. IL-6 may function as a critical anti-apoptotic factor in the liver by its ability to establish and maintain an adequate level of FLIP and downstream anti-apoptotic factors.

Liver regeneration following 70% partial hepatectomy leads to rapid activation of genes in the remnant liver. Interleukin-6 deficient (IL-6 -/-) mice have impaired liver regeneration and abnormalities in immediate early gene expression. In this study, the gene expression program in the IL-6 +/+ and -/- livers at 2 hours posthepatectomy was examined with a cDNA array representing 588 highly regulated mouse genes. Thirty-six percent of the 103 immediate early genes were induced differently in IL-6 +/+ compared with IL-6 -/- livers, implying regulation by IL-6. IL-6 treatment of the IL-6 -/- mice in the absence of hepatectomy induced a much smaller set of genes in the liver, suggesting that IL-6 cooperates with other hepatectomy-induced factors to activate the large number of genes. Northern blot analyses were used to verify gene expression data obtained from the arrays. The expression of urokinase type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), critical components of the urokinase plasminogen activator (uPA) system, was lower and delayed in IL-6 -/- livers. Despite the fact that active uPAR/uPA complex is critical for hepatocyte growth factor (HGF) activation, no differences were detected between the IL-6 +/+ and -/- livers in HGF activation as measured by receptor phosphorylation. On the contrary, the mitogen-activated protein kinase (MAPK) pathway was activated in IL-6 +/+ livers early during regeneration but remarkably delayed in IL-6 -/- livers. Defective liver regeneration may be explained by the large number of gene activation pathways altered in IL-6 -/- livers and further supports the finding that IL-6 is necessary for normal liver regeneration.

Following hepatic injury or stress, gluconeogenic and acute-phase response genes are rapidly upregulated to restore metabolic homeostasis and limit tissue damage. Regulation of the liver-restricted insulin-like growth factor binding protein 1 (IGFBP-1) gene is dramatically altered by changes in the metabolic state and hepatectomy, and thus it provided an appropriate reporter to assess the transcriptional milieu in the liver during repair and regeneration. The cytokine interleukin-6 (IL-6) is required for liver regeneration and repair, and it transcriptionally upregulates a vast array of genes during liver growth by unknown mechanisms. Evidence for a biologic role of IL-6 in IGFBP-1 upregulation was demonstrated by increased expression of hepatic IGFBP-1 in IL-6 transgenic and following injection of IL-6 into nonfasting animals and its reduced expression in IL-6(-/-) livers posthepatectomy. In both hepatic and nonhepatic cells, IL-6 -mediated IGFBP-1 promoter activation was via an intact hepatocyte nuclear factor 1 (HNF-1) site and was dependent on the presence of endogenous liver factor HNF-1 and induced factors STAT3 and AP-1 (c-Fos/c-Jun). IL-6 acted through the STAT3 pathway, as dominant negative STAT3 completely blocked IL-6-mediated stimulation of the IGFBP-1 promoter via the HNF-1 site. HNF-1/c-Fos and HNF-1/STAT3 protein complexes were detected in mouse livers and in hepatic and nonhepatic cell lines overexpressing STAT3/c-Fos/HNF-1. Similar regulation was demonstrated using glucose-6-phosphatase and alpha-fibrinogen promoters, indicating that HNF-1/IL-6/STAT3/AP-1-mediated transactivation of hepatic gene expression is a general phenomenon after liver injury. These results demonstrate that the two classes of transcription factors, growth induced (STAT3 and AP-1) and tissue specific (HNF-1), can interact as an adaptive response to liver injury to amplify expression of hepatic genes important for the homeostatic response during organ repair.

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.

Mutations in Jagged1, a Notch ligand, have been shown to result in Alagille syndrome (AGS), however, the causal link between haploinsufficiency of Jagged1 and intrahepatic ductal paucity is unknown. This survey was performed to determine the expression pattern of Jagged1 in the fetal and postnatal liver. Reverse transcription polymerase chain reaction (RT-PCR) showed Jagged1 expression in all samples studied including rat liver embryonic days 16 to 21, 1-day-old, 1-week-old, and 2-month-old adult rats. RT-PCR detected Jagged1 in total liver RNA extracted from cadaver organ donor samples from reduced human grafts and explanted native livers from a variety of pediatric disorders including AGS, biliary atresia, congenital hepatic fibrosis, sclerosing cholangitis, cystic fibrosis, fulminant hepatic failure, tyrosinemia, and chronic rejection. Immunohistochemistry showed Jagged1 expression in human fetal samples localized to the ductal plate from 14-week gestation onward. Expression in the postnatal liver was seen in biliary epithelium and zone 3 hepatocytes. In conclusion, these studies show that Jagged1 is expressed in the fetal and postnatal liver in health and disease. We show localization of expression by immunohistochemistry to ductal plate epithelium in human fetal samples and to the biliary epithelium and zone 3 hepatocytes in human postnatal samples. Our results show the localization of Jagged1 in fetal liver and demonstration of Jagged1 expression in postnatal rat and human liver specimens. Further studies of Jagged1 and the Notch signaling pathway are expected to elucidate mechanisms of the regulation of biliary epithelial growth and development.

Studies with tumor necrosis factor p55 receptor- and interleukin-6 (IL-6)-deficient mice have shown that IL-6 is required for hepatocyte proliferation and reconstitution of the liver mass after partial hepatectomy. The biological activities of IL-6 are potentiated when this cytokine binds soluble forms of its specific receptor subunit (sIL-6R) and the resulting complex interacts with the transmembrane signaling chain gp130. We show here that double transgenic mice expressing high levels of both human IL-6 and sIL-6R under the control of liver-specific promoters spontaneously develop nodules of hepatocellular hyperplasia around periportal spaces and present signs of sustained hepatocyte proliferation. The resulting picture is identical to that of human nodular regenerative hyperplasia, a condition frequently associated with immunological and myeloproliferative disorders. In high expressors, hyperplastic lesions progress with time into discrete liver adenomas. These data strongly suggest that the IL-6/sIL-6R complex is both a primary stimulus to hepatocyte proliferation and a pathogenic factor of hepatocellular transformation.

Hepatic gene expression of P-enolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (Glc-6-Pase) is regulated in response to changes in the availability of substrates, in particular glucose (Glc; Massillon, D., Barzilai, N., Chen, W., Hu, M., and Rossetti, L. (1996) J. Biol. Chem. 271, 9871-9874). We investigated the mechanism(s) in conscious rats. Hyperglycemia per se caused a rapid and marked increase in Glc-6-Pase mRNA abundance and protein levels. By contrast, hyperglycemia decreased the abundance of PEPCK mRNA. Importantly, inhibition of glucokinase activity by glucosamine infusion blunted both the stimulation of Glc-6-Pase and the inhibition of PEPCK gene expression by Glc, suggesting that an intrahepatic signal (metabolite) generated by the metabolism of glucose at or beyond Glc-6-P was responsible for the regulatory effect of Glc. The effect of Glc on the L-type pyruvate kinase gene is mediated by xylulose-5-P (Doiron, B., Cuif, M., Chen, R., and Kahn, A. (1996) J. Biol. Chem. 271, 5321-5324). Thus, we next investigated whether an isolated increase in the hepatic concentration of this metabolite can also reproduce the effects of Glc on Glc-6-Pase and PEPCK gene expression in vivo. Xylitol, which is directly converted to xylulose-5-P in the liver, was infused to raise the hepatic concentration of xylulose-5-P by approximately 3-fold. Xylitol infusion did not alter the levels of Glc-6-P and of fructose-2,6-biphosphate. However, it replicated the effects of hyperglycemia on Glc-6-Pase and PEPCK gene expression and resulted in a 75% increase in the in vivo flux through Glc-6-Pase (total glucose output).

Sepsis in rats decreases the hepatic expression of the gluconeogenic enzyme glucose-6-phosphatase (G6Pase). The aim of this study was to investigate the relationship among G6Pase transcription, mRNA, enzymatic activity, and serum glucose levels at different intervals during mild or fulminant sepsis. Both fulminant and mild sepsis immediately decreased hepatic G6Pase mRNA levels. In mild sepsis, levels began to recover late in the time course. Serum glucose levels were maintained in mild sepsis but decreased markedly in fulminant sepsis. G6Pase transcription after fulminant sepsis decreased and never recovered. A similar transcriptional decrease was noted in mild sepsis, but some recovery occurred in this state. Histochemistry after mild sepsis revealed a decrease in G6Pase protein and enzymatic activity that paralleled transcription. These studies suggest that changes in G6Pase transcription and activity are early markers for sepsis-induced alterations in hepatic function. Mechanisms other than gene expression and enzymatic activity serve to maintain glucose levels in mild sepsis, but in the fulminant disorder, compensatory mechanisms fail and hypoglycemia develops.

Rat CL-6 is the most highly insulin-induced gene in a liver cell line and is expressed in proliferating liver during regeneration and development. CL-6 is now denoted INSIG1 (insulin-induced gene 1). Human INSIG1 was isolated and found to be 80% identical to the rat gene within the translated region. It was located on human chromosome 7 within band q36. The human INSIG1 promoter conferred a high level of expression in both liver and fibroblast cell lines. INSIG1 expression was upregulated at the transcriptional level in rat regenerating liver and induced in a model of murine adipocyte differentiation, suggesting that INSIG1 may play a role in growth and differentiation of tissues involved in metabolic control.

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.

Hepatocellular dysfunction in sepsis may be neutrophil mediated. We therefore tested the hypothesis that sepsis-induced neutrophil accumulation is associated with increased expression of the chemokine, cytokine-induced neutrophil chemoattractant (CINC). In Sprague-Dawley rats made septic by cecal ligation and puncture, we demonstrate a time-dependent increase in CINC mRNA, which returns to baseline by 48 h. By in situ hybridization, this mRNA is present in hepatocytes and nonparenchymal cells. CINC protein levels in septic animals parallel mRNA levels and resolve by 48 h. Because CINC expression is induced by cytokines including tumor necrosis factor-alpha (TNF- alpha), we show, by immunohistochemistry, that sepsis elevates intrahepatic TNF-alpha. Finally, because the CINC promoter is transactivated by the transcription factor, nuclear factor kappa B (NF-kappa B), we determined that hepatic NF-kappa B DNA binding increases dramatically, peaking 16 h after cecal ligation and puncture. Thus activated NF-kappa B may mediate CINC induction in sepsis. This constellation of findings suggests a mechanism by which sepsis may induce neutrophil accumulation in the liver and may have implications regarding sepsis-induced hepatic dysfunction.

Patients with hereditary tyrosinemia type 1 have a deficiency of fumarylacetoacetate hydrolase (FAH) and develop progressive hepatocellular dysfunction with a high risk of malignant transformation. Serum alpha-fetoprotein levels are frequently elevated in these patients; therefore, this commonly used marker of tumorigenesis is inadequate. To date, no literature exists describing the hepatic gene alterations in patients with this disease. We analyzed the expression of a panel of proliferation associated and liver-specific genes in the liver of a 33 month-old girl at the time of orthotopic liver transplantation. This study provides information that may be useful in developing markers for malignancy and understanding the pathogenesis of this disease. Gene expression patterns of two regenerating nodules and total liver from the patient with FAH deficiency were compared with control donor liver. Liver-specific and growth-induced genes with altered expression in the tyrosinemic liver included several functional classes: structural proteins (actin, thrombospondin), transcription factors (c-fos, egr-1, C/EBPalpha), liver-specific enzymes (glucose-6-phosphatase [G6Phase], and secreted factors (insulin-like growth factor binding protein 1 [GFBP-1]. Isolated macronodules demonstrated varied patterns of expression, suggesting that they do not form a homogeneous cellular environment. In the tyrosinemic liver, IGFBP-1 messenger RNA expression was high and G6Phase messenger RNA was not detectable. Although G6Phase and IGFBP-1 are coexpressed in regenerating liver, immunohistochemistry in the tyrosinemic liver demonstrated a mutually exclusive distribution for the two proteins in a tissue section with features of dysplasia. We propose that cells in these areas may have an aberrant transcription factor and growth factor "milieu" that leads to altered gene and protein expression. These molecular alterations are reflected in dysplastic histologic changes and may ultimately predispose to the development of malignancy.

Mechanisms controlling the tyrosine phosphorylation of cellular proteins are important in the regulation of cellular processes including growth and differentiation. It has become clear that a number of protein tyrosine phosphatases (PTPases) that dephosphorylate tyrosyl residues may play a role in the growth response, both in growth-promoting and growth-inhibiting capacities. We identified PRL-1, a unique nuclear PTPase that is an immediate-early gene in liver regeneration and is positively associated with growth, including fetal and neoplastic hepatic growth and anchorage-independent growth after overexpression in fibroblasts. In this study, we show that PRL-1 nuclear protein levels in regenerating liver parallel those of its mRNA, although the peak occurs later, just before the onset of DNA synthesis. We further show that PRL-1 is significantly expressed in intestinal epithelia and that, in contrast to the expression pattern of PRL-1 in liver, its expression is associated with cellular differentiation in intestine. Specifically, PRL-1 is expressed in villus but not crypt enterocytes and in confluent differentiated but not undifferentiated proliferating Caco-2 colon carcinoma cells. The expression of PRL-1 in intestine shows inverse correlation with proliferating cell nuclear antigen expression, a marker for S-phase cells. These results suggest that PRL-1 may play different roles in these two digestive tissues. Such a dichotomy of roles has previously been described for some protein tyrosine kinases and might be due to the availability of alternate substrates in different tissues.

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)

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.

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 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.