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.

Sepsis-induced depression of rat glucose-6-phosphatase gene expression and activity.

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.

HRS/SRp40-mediated inclusion of the fibronectin EIIIB exon, a possible cause of increased EIIIB expression in proliferating liver.

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.

Increased expression of cytokine-induced neutrophil chemoattractant in septic rat liver.

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.

Expression of PRL-1 nuclear PTPase is associated with proliferation in liver but with differentiation in intestine.

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.

Variable gene expression within human tyrosinemia type 1 liver may reflect region-specific dysplasia.

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.

Coexistence of C/EBP alpha, beta, growth-induced proteins and DNA synthesis in hepatocytes during liver regeneration. Implications for maintenance of the differentiated state during liver growth.

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.

High levels of glucose-6-phosphatase gene and protein expression reflect an adaptive response in proliferating liver and diabetes.

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.

Rapid activation of post-hepatectomy factor/nuclear factor kappa B in hepatocytes, a primary response in the regenerating liver.

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.

Structure and localization of the IGFBP-1 gene and its expression during liver regeneration.

Insulin-like growth factor-binding protein-1s are important modulators of the insulin-like growth factors that may have both positive and negative effects on the ability of insulin-like growth factors to stimulate cell growth. The IGFBP-1 gene is one of the most highly induced immediate-early genes after partial hepatectomy. The IGFBP-1 gene is also expressed at a high level during fetal liver development and in response to nutritional changes and diabetes. Therefore it may have important roles in liver growth and metabolism. To begin to examine the regulation of this gene, we cloned and sequenced the entire mouse IGFBP-1 gene. Its structure is highly similar to that of the human gene, and, in addition to the exonic regions, the two genes are highly conserved in specific regions in the promoter and first intron. Analysis of this conservation allows us to predict important regulatory sites that define the tissue specific and insulin-mediated regulation of the gene and identify potential sites that might be important for the transcriptional induction during liver regeneration. The mouse gene is located on mouse chromosome 11; it is found at the boundary between regions in the mouse genome homologous to human chromosomes 22 and 7. We found IGFBP-1 mRNA in both parenchymal and nonparenchymal RNA after partial hepatectomy. Using in situ hybridization of IGFBP-1 mRNA in regenerating rat liver tissue, we demonstrated IGFBP-1 transcripts in several cell types. We found that IGFBP-1 gene induction after partial hepatectomy is paralleled by protein expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel delayed-early and highly insulin-induced growth response genes. Identification of HRS, a potential regulator of alternative pre-mRNA splicing.

We have identified 41 novel and many previously known growth response genes induced in regenerating liver and insulin-treated Reuber H35 cells, a rat hepatoma cell line that grows in response to physiologic concentrations of insulin and retains some properties of regenerating liver. Although many genes are expressed similarly in the two systems, there are important differences in the kinetics of induction of some genes. These differences allowed us to identify and characterize novel genes that are highly insulin-induced and expressed as delayed-early genes in regenerating liver. Sequence analysis of CL-6, the most abundant insulin-induced gene, resulted in the identification of a highly hydrophobic hepatic protein. Sequence analysis of HRS, a highly insulin-induced delayed-early gene, demonstrated that it is a member of the family of regulators of alternative pre-mRNA splicing. Different forms of HRS mRNA are temporally regulated during the growth response, suggesting that HRS could autoregulate processing of its pre-mRNA. Given the dramatic increase in RNA production during late G1, proteins induced by mitogens like insulin that control RNA processing are likely to have important roles in cell cycle regulation.

Induction patterns of 70 genes during nine days after hepatectomy define the temporal course of 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.

Cholestatic jaundice in the newborn.

Neonatal cholestasis or direct hyperbilirubinemia in the first 3 months of life is nearly always more pathologic than physiologic. This article reviews the extensive differential diagnosis of cholestatic jaundice and the approach to diagnosis. A sense of urgency is underscored by the value of early recognition and intervention in metabolic, iatrogenic, and anatomic disorders. The long-term management goals are reviewed, including indications for hepatic transplantation.