Two-stage operative cytoreduction and intraperitoneal chemotherapy for diffuse malignant peritoneal mesothelioma: Operative morbidity and mortality in phase I and II trials.

AIMS: The standard of care for diffuse malignant peritoneal mesothelioma involves operative cytoreduction and intraperitoneal chemotherapy. Most centers favor aggressive operative cytoreduction, accepting high morbidity and mortality. In our trials, patients underwent less extensive cytoreduction followed by prolonged intraperitoneal chemotherapy. Patients underwent a second cytoreduction with heated intraperitoneal chemotherapy. We hypothesized this would result in lower operative morbidity and mortality with similar survival. METHODS: Hospital records, discharge summaries, microbiology, radiography, and office records were retrospectively reviewed to supplement a prospective database. 30-day morbidity and mortality were categorized, and classified according to the Clavien methodology. RESULTS: 47 first and 39 second operations were performed with 13% and 26% morbidity, respectively. Mortality was 2%. Infections comprised 59% of the morbidity. Inclusive of both operations, formal peritonectomy was performed in 16% of patients, resection of isolated lesions in less than half, and only 19% had a visceral organs other than the spleen resected. At the completion of the protocol, only 3% of patients had visible intraperitoneal disease. The mean total length of stay for both operations combined was 16 ± 23 days. Overall median survival was 54.9 months, and median survival for the epithelioid subtype was 70.2 months. CONCLUSIONS: A two-stage cytoreduction with intraperitoneal chemotherapy offers median survival comparable to one-stage protocols, with relatively low morbidity, mortality, visceral resections and length of stay despite two operations. This series supports that our protocol is a feasible and safe approach.

Molecular profiling of malignant peritoneal mesothelioma identifies the ubiquitin-proteasome pathway as a therapeutic target in poor prognosis tumors.

Malignant mesothelioma is an aggressive neoplastic proliferation derived from cells lining serosal membranes. The biological and clinical characteristics of epithelial type malignant mesothelioma are distinct from those of biphasic and sarcomatous type tumors. The goal of our study was to examine the molecular basis for this distinction. Microarray analysis confirmed that the molecular signatures of epithelial and biphasic histologic subtypes were distinct. Among the differentially expressed functional gene categories was the ubiquitin-proteasome pathway, which was upregulated in biphasic tumors. Cytotoxicity experiments indicated that 211H cells derived from biphasic tumors were synergistically sensitive to sequential combination regimens containing the proteasome inhibitor bortezomib and oxaliplatin. The mechanism of this synergistic response, which was not detected in cells of epithelial tumor origin, was apoptosis. Together, our results identify the ubiquitin-proteasome pathway as a biomarker of poor prognosis biphasic peritoneal mesothelioma tumors and suggest that proteasome inhibitors could increase the effectiveness of cytotoxic chemotherapy in this subset of patients.

Current treatment options and biology of peritoneal mesothelioma: meeting summary of the first NIH peritoneal mesothelioma conference.

Peritoneal mesothelioma is a rare cancer of the peritoneum with about 250 new cases diagnosed each year in the United States. It is the second most common site for mesothelioma development and accounts for 10-20% of all mesotheliomas diagnosed in the United States. A meeting sponsored by the NIH Office of Rare Diseases was held in Bethesda, Maryland on September 13 and 14, 2004. The objective of this meeting was to review the epidemiology, biology and current surgical and medical management of peritoneal mesothelioma. In addition, the meeting also discussed clinical and pre-clinical evaluation of novel treatments for mesothelioma as well as ongoing laboratory research to better understand this disease. This report summarizes the proceedings of the meeting as well as directions for future clinical and basic research.

Global changes in interleukin-6-dependent gene expression patterns in mouse livers after partial hepatectomy.

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.

Interleukin-6 protects against Fas-mediated death by establishing a critical level of anti-apoptotic hepatic proteins FLIP, Bcl-2, and Bcl-xL.

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.

Normal liver regeneration in p50/nuclear factor kappaB1 knockout mice.

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.

Activation of interleukin-6/STAT3 and liver regeneration following transplantation.

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.

ATF-7, a novel bZIP protein, interacts with the PRL-1 protein-tyrosine phosphatase.

We have identified a novel basic leucine zipper (bZIP) protein, designated ATF-7, that physically interacts with the PRL-1 protein-tyrosine phosphatase (PTPase). PRL-1 is a predominantly nuclear, farnesylated PTPase that has been linked to the control of cellular growth and differentiation. This interaction was initially found using the yeast two-hybrid system. ATF-7 is most closely related to members of the ATF/CREB family of bZIP proteins, with highest homology to ATF-4. ATF-7 homodimers can bind specifically to CRE elements. ATF-7 is expressed in a number of different tissues and is expressed in association with differentiation in the Caco-2 cell model of intestinal differentiation. We have confirmed the PRL-1.ATF-7 interaction and mapped the regions of ATF-7 and PRL-1 important for interaction to ATF-7's bZIP region and PRL-1's phosphatase domain. Finally, we have determined that PRL-1 is able to dephosphorylate ATF-7 in vitro. Further insight into ATF-7's precise cellular roles, transcriptional function, and downstream targets are likely be of importance in understanding the mechanisms underlying the complex processes of maintenance, differentiation, and turnover of epithelial tissues.

Interleukin-6-induced STAT3 and AP-1 amplify hepatocyte nuclear factor 1-mediated transactivation of hepatic genes, an adaptive response to liver injury.

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.

Expression of the protein tyrosine phosphatase, phosphatase of regenerating liver 1, in the outer segments of primate cone photoreceptors.

Foveal cone photoreceptors are morphologically distinct and, presumably, express unique transcripts. We have identified a cDNA clone encoding the protein tyrosine phosphatase (PTP), phosphatase of regenerating liver 1 (PRL-1) in a screen for genes that are enriched in monkey fovea. PRL-1 was originally isolated as an immediate early gene in regenerating liver [R.H. Diamond, D.E. Cressman, T.M. Laz, C.S. Abrams, R. Taub, PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth, Mol. Cell Biol. 14 (1994) 3752-3762]. On cDNA Southern blots of human and monkey retina, radiolabeled PRL-1 cDNA hybridized to a single mRNA species of about 2.5 kb that was most intense in fovea-enriched samples. The monkey PRL-1 deduced amino acid sequence is identical to human, rat and mouse PRL-1. Affinity-purified antibodies directed against PRL-1 preferentially labeled cone photoreceptor cells and a subpopulation of bipolar cells in monkey retina. Immunoreactivity in cones was confined to red and green, but not to blue, cones and was restricted to the outer segments. Immunolocalization also revealed that PRL-1 protein expression was non-nuclear, suggesting that its function in the retina may be unrelated to its role in other tissues where it is expressed primarily in nuclei. Although both foveal and extrafoveal cones were PRL-1 reactive, the high abundance of PRL-1 mRNAs detected in monkey fovea correlates with the high concentration of cones in the fovea. The PRL-1 gene is located on chromosome 6q within an interval that also contains the genes that cause two hereditary retinal dystrophies. These studies demonstrate novel expression of the PRL-1 gene in the neural retina and suggest the phosphatase activity of PRL-1 may modulate normal cone photoreceptor cell function.

Effects of flecainide in patients with new SCN5A mutation: mutation-specific therapy for long-QT syndrome?

BACKGROUND: Mutations in the cardiac sodium channel gene (SCN5A) can cause one variant of the congenital long-QT syndrome. The effects of some of these mutations on the alpha-subunit channel properties can be blocked by type Ib antiarrhythmic drugs. Recently, we have described a new SCN5A mutation (D1790G) that affects the channel properties in a manner suggesting that sodium blockers of the Ib type will be ineffective in carriers of this mutation. Hence, the ECG effects of flecainide-acetate, a type Ic sodium blocker, were evaluated in carriers of this mutation. METHODS AND RESULTS: Eight asymptomatic mutation carriers and 5 control subjects were studied. Intravenous lidocaine was tested first in only 2 mutation carriers and had no significant effect on any ECG parameter. Flecainide significantly shortened all heart rate-corrected repolarization duration parameters only in carriers and not in control subjects: QT(c) shortened by 9.5% (from 517+/-45 to 468+/-36 ms, P=0.011), and the S-offset to T-onset interval shortened by 64.7% (from 187+/-88 to 66+/-50 ms, P=0.0092). Flecainide also normalized the marked baseline repolarization dispersion in most mutation carriers. These effects among carriers were maintained during long-term (9 to 17 months) outpatient flecainide therapy with no adverse effects. CONCLUSIONS: This report is the first to describe SCN5A mutation carriers who significantly responded to flecainide therapy yet did not respond to lidocaine. These results have important implications for long-QT allele-specific therapeutic strategies.

Peritoneal mesothelioma.

Malignant peritoneal mesothelioma is an aggressive neoplasm that rapidly spreads within the confines of the abdominal cavity to involve most accessible peritoneal and omental surfaces. Current treatments are unsatisfactory, and new approaches are needed. We have noted prolonged survival in selected patients after intensive multimodality treatment. Our current experimental regimen includes initial laparotomy with omentectomy, resection of peritoneal implants, and placement of bilateral peritoneal Port-a-Caths (Sims Deltec, Inc., St. Paul, MN); repeated courses of intraperitoneal chemotherapy with doxorubicin, cisplatin, and interferon gamma; second-look laparotomy and intraoperative hyperthermic perfusion with mitomycin and cisplatin; and whole abdominal radiation. Patients with peritoneal mesothelioma who are not candidates for this approach can sometimes be palliated with systemic (intravenous) chemotherapy using doxorubicin or mitomycin, alone or in combination with cisplatin or carboplatin. Newer agents such as gemcitabine and multitargeted antifolate (pemetrexed disodium, LY231514) show promise of greater effectiveness.

Increased toxin-induced liver injury and fibrosis in interleukin-6-deficient mice.

Interleukin-6 null (IL-6-/-) mice have impaired liver regeneration and increased liver necrosis following partial hepatectomy that is corrected with IL-6 treatment. Following acute carbon tetrachloride (CCl(4)) treatment, we found that IL-6-/- mice developed increased hepatocellular injury and defective regeneration with significant blunting of signal transducer-and-activator of transcription protein 3 (STAT3) and nuclear factor-kappaB (NF-kappaB) activation and reduced hepatocyte DNA synthetic and mitotic responses. After CCl(4) treatment, unlike partial hepatectomy, increased hepatocyte apoptosis was noted in IL-6-/- livers. Pretreatment with IL-6 before CCl(4) reduced acute CCl(4) injury and apoptosis and accelerated regeneration in both IL-6+/+ and -/- livers. Repetitive doses of CCl(4) in the presence or absence of phenobarbital resulted in increased injury and fibrosis in IL-6 -/- compared with +/+ livers. After acute and chronic injury, IL-6-/- livers showed the protracted presence of alpha-smooth muscle actin associated with activated stellate cells, indicating a disturbed response in wound healing that progressed to fibrosis. These data provide evidence for an important role for IL-6 in reducing CCl(4)-induced acute and chronic liver injury and fibrosis.

Liver-specific and proliferation-induced deoxyribonuclease I hypersensitive sites in the mouse insulin-like growth factor binding protein-1 gene.

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.

Hepatic jagged1 expression studies.

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.

Transcriptional regulatory signals define cytokine-dependent and -independent pathways in liver regeneration.

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.

Mitogenic up-regulation of the PRL-1 protein-tyrosine phosphatase gene by Egr-1. Egr-1 activation is an early event in liver regeneration.

The cellular signals that initiate cell growth are incompletely understood. Insight could be provided by understanding the signals regulating the transcriptional induction of immediate-early genes which occurs within minutes of the growth stimulus. The expression of the PRL-1 gene, which encodes a unique nuclear protein-tyrosine phosphatase, is rapidly induced in regenerating liver and mitogen-treated cells. Transcription of the PRL-1 gene increased in the rat liver remnant within a few minutes after partial hepatectomy and largely explained the increase in steady-state PRL-1 mRNA in the first few hours posthepatectomy. Egr-1 (early growth response factor) specifically bound a region of the proximal PRL-1 promoter P1 (-99). Egr-1 binding activity was more rapidly induced in regenerating liver than mitogen-treated H35 and NIH 3T3 cells, remained elevated through 4 h posthepatectomy, and appeared to be dependent not only on new Egr-1 protein synthesis but on post-translational regulation of Egr-1. Egr-1 efficiently transactivated a PRL-1 promoter reporter construct containing an intact not mutant Egr-1 site, and the Egr-1 site largely accounted for PRL-1 gene up-regulation in response to mitogen stimulation. These data predict that Egr-1 activation is an early event in liver regeneration and mitogen-activated cells that provides a regulatory stimulus for a subset of immediate-early genes.

Transcriptional up-regulation of the delayed early gene HRS/SRp40 during liver regeneration. Interactions among YY1, GA-binding proteins, and mitogenic signals.

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.

Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver.

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.