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Intrahepatic cholangiocarcinoma (ICC) is the second most common primary malignancy in the liver. ICC has been classified as a malignant tumor arising from cholangiocytes; however, the co-occurrence of ICC and viral hepatitis suggests that ICC originates in hepatocytes. In order to determine the cellular origin of ICC, we used a mouse model of ICC in which hepatocytes and cholangiocytes were labeled with heritable, cell type-specific reporters. Our studies reveal that ICC is generated by biliary lineage cells derived from hepatocytes, rather than cholangiocytes. Additionally, we found that Notch activation is critical for hepatocyte conversion into biliary lineage cells during the onset of ICC and its subsequent malignancy and progression. These findings will help to elucidate the pathogenic mechanism of ICC and to develop therapeutic strategies for this refractory disease., Introduction ICC is the second most prevalent primary malignancy in the liver, and its incidence and mortality rates are increasing worldwide (1), (2). Intrahepatic cholangiocarcinoma (ICC) has been known as [...]

Over half of the mature hepatocytes in mice and humans are aneuploid and yet retain full ability to undergo mitosis. This observation has raised the question of whether this unusual somatic genetic variation evolved as an adaptive mechanism in response to hepatic injury. According to this model, hepatotoxic insults select for hepatocytes with specific numerical chromosome abnormalities, rendering them differentially resistant to injury. To test this hypothesis, we utilized a strain of mice heterozygous for a mutation in the homogentisic acid dioxygenase (Hgd) gene located on chromosome 16. Loss of the remaining Hgd allele protects from fumarylacetoacetate hydrolase (Fah) deficiency, a genetic liver disease model. When adult mice heterozygous for Hgd and lacking Fah were exposed to chronic liver damage, injury-resistant nodules consisting of Hgdnull hepatocytes rapidly emerged. To determine whether aneuploidy played a role in this phenomenon, array comparative genomic hybridization (aCGH) and metaphase karyotyping were performed. Strikingly, loss of chromosome 16 was dramatically enriched in all mice that became completely resistant to tyrosinemia-induced hepatic injury. The frequency of chromosome 16-specific aneuploidy was approximately 50%. This result indicates that selection of a specific aneuploid karyotype can result in the adaptation of hepatocytes to chronic liver injury. The extent to which aneuploidy promotes hepatic adaptation in humans remains under investigation., Introduction Since all portal blood first passes through the liver, this organ is continuously exposed to nutritional challenges as well as xenobiotic insults (reviewed in refs. 1, 2). As a [...]

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system., What are the molecular mechanisms underlying the biogenesis, number and size of cellular organelles? Organelles of the biosynthetic and endocytic pathway possess a toolbox of coat components, Rab GTPases, SNARE [...]

Etiologic risk factors for hepatocellular carcinoma can be involved in the transformation process by directly targeting intracellular signaling pathways or by indirectly stimulating chronic cycles of hepatocyte destruction and regeneration. However, the contribution of these two routes to hepatocarcinogenesis has not been determined, partly because of the difficulty in distinguishing damaged and regenerated hepatocytes. Here we report that induced deletion of the damaged DNA binding protein 1 (DDB1) abrogates the self-renewing capacity of hepatocytes, resulting in compensatory proliferation of DDB1-expressing hepatocytes. Constitutive stimulation of this regeneration process leads to development of hepatocellular carcinoma, which surprisingly contains no disruption of the DDB1 gene, indicating a cell-nonautonomous role of DDB1 inactivation in tumor initiation. Our results suggest that viruses and hepatoxins may cause liver tumors by simply driving hepatocyte turnover without directly targeting cancer cells. ubiquitin ligase | mouse model | gene knockout doi/ 10.1073/pnas.1015793108

Having successfully developed mechanisms to evade immune clearance, hepatitis C virus (HCV) establishes persistent infection in approximately 75%-80% of patients. In these individuals, the function of HCV-specific [CD8.sup.+] T cells is impaired by ligation of inhibitory receptors, the repertoire of which has expanded considerably in the past few years. We hypothesized that the coexpression of the negative regulatory receptors T cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) and programmed death 1 (PD-1) in HCV infection would identify patients at risk of developing viral persistence during and after acute HCV infection. The frequency of PD-[1.sup.-]Tim-[3.sup.-] HCV-specific CTLs greatly outnumbered [PD-1.sup.+][Tim-3.sup.+] CTLs in patients with acute resolving infection. Moreover, the population of [PD-1.sup.+][Tim-3.sup.+] T cells was enriched for within the central memory T cell subset and within the liver. Blockade of either PD-1 or Tim-3 enhanced in vitro proliferation of HCV-specific CTLs to a similar extent, whereas cytotoxicity against a hepatocyte cell line that expressed cognate HCV epitopes was increased exclusively by Tim-3 blockade. These results indicate that the coexpression of these inhibitory molecules tracks with defective T cell responses and that anatomical differences might account for lack of immune control of persistent pathogens, which suggests their manipulation may represent a rational target for novel immunotherapeutic approaches., Introduction Chronic viral infections, such as those caused by HCV, HBV, and HIV, are among the leading causes of morbidity and mortality in the world (1). These viruses have successfully [...]

Although multiple determinants for hepatitis C virus (HCV) infection are known, it remains partly unclear what determines the human specificity of HCV infection. Presumably, the presence of appropriate entry receptors is essential, and this may explain why HCV is unable to infect nonhuman hepatocytes. However, using mice with chimeric human livers, we show in this study that the presence of human hepatocytes, and therefore human entry receptors, is not sufficient for HCV infection. In successfully transplanted SCID/Alb-uPA mice, infection with HCV is reliable only when ~70-80% of the liver consists of human hepatocytes. We show that chimeric mice, which are hard to infect with HCV, have significant groups of human hepatocytes that are readily infected with hepatitis B virus. Thus it is unlikely that the lack of infection with HCV can simply be attributed to low hepatocyte numbers. We investigated whether the humanization of lipoprotein profiles is positively associated with infection success. We show that the lipoprotein profiles of chimeric mice become more human-like at high levels of engraftment of human hepatocytes. This and expression of markers of human lipoprotein biosynthesis, human apolipoprotein B (ApoB) and cholesterol ester transfer protein (CETP), show a strong positive correlation with successful infection. Association of HCV in the blood of chimeric mice to ApoB-containing lipoproteins is comparable to association of HCV in patient serum and provides further support for a critical role for ApoB-containing lipoproteins in the infectious cycle of HCV. Our data suggest that the weakest link in the HCV infection chain does not appear to be the presence of human hepatocytes per se. We believe that HCV infection also depends on the presence of sufficient levels of human lipoproteins. apolipoprotein B; low-density lipoprotein; xenotransplantation; immunodeficient mouse; Flavivirus doi: 10.1152/ajpgi.00200.2010.

Chronic alcohol exposure has been shown to increase the gut permeability in the distal intestine, in part, through induction of zinc deficiency. The present study evaluated the molecular mechanisms whereby zinc deficiency mediates alcohol-induced intestinal barrier dysfunction. Examination of zinc finger transcription factors in the gastrointestinal tract of mice revealed a prominent distribution of hepatocyte nuclear factor-4[alpha] (HNF-4[alpha]). HNF-4[alpha] exclusively localizes in the epithelial nuclei and exhibited an increased abundance in mRNA and protein levels in the distal intestine. Chronic alcohol exposure to mice repressed the HNF-4[alpha] gene expression in the ileum and reduced the protein level and DNA binding activity of HNF-4[alpha] in all of the intestinal segments with the most remarkable changes in the ileum. Chronic alcohol exposure also decreased the mRNA levels of tight junction proteins, particularly in the ileum. Caco-2 cell culture studies were conducted to determine the role of HNF-4[alpha] in regulation of the epithelial tight junction and barrier function. Knockdown of HNF-4[alpha] in Caco-2 cells decreased the mRNA and protein levels of tight junction proteins in association with disruption of the epithelial barrier. Alcohol treatment inactivated HNF-4[alpha], which was prevented by N-acetyl-cysteine or zinc. The link between zinc and HNF-4[alpha] function was confirmed by zinc deprivation, which inhibited HNF-4[alpha] DNA binding activity. These results indicate that inactivation of HNF-4[alpha] due to oxidative stress and zinc deficiency is likely a novel mechanism contributing to the deleterious effects of alcohol on the tight junctions and the intestinal barrier function. zinc deprivation; oxidative stress; claudin-1; occludin; zonula occludens-1 doi: 10.1152/ajpgi.00515.2009.

The cell death receptor Fas plays a role in the establishment of fulminant hepatitis, a major cause of druginduced liver failure. Fas activation elicits extrinsic apoptotic and hepatoprotective signals; however, the mechanisms by which these signals are integrated during disease are unknown. Tissue inhibitor of metalloproteinases 3 (TIMP3) controls the critical sheddase a disintegrin and metalloproteinase 17 (ADAM17) and may dictate stress signaling. Using mice and cells lacking TIMP3, ADAM17, and ADAM17-regulated cell surface molecules, we have found that ADAM17-mediated ectodomain shedding of TNF receptors and EGF family ligands controls activation of multiple signaling cascades in Fas-induced hepatitis. We demonstrated that TNF signaling promoted hepatotoxicity, while excessive TNF receptor 1 (TNFR1) shedding in [Timp3.sup.-/-] mice was protective. Compound [Timp3.sup.-/-][Tnf.sup.-/-] and [Timp3.sup.-/-] [Tnfr1.sup.-/-] knockout conferred complete resistance to Fas-induced toxicity. Loss of Timp3 enhanced metalloproteinase-dependent EGFR signaling due to increased release of the EGFR ligands TGF-α, amphiregulin, and HB-EGF, while depletion of shed amphiregulin resensitized [Timp3.sup.-/-] hepatocytes to apoptosis. Finally, adenoviral delivery of Adam17 prevented acetaminophen-induced liver failure in a clinically relevant model of Fas-dependent fulminant hepatitis. These findings demonstrate that TIMP3 and ADAM17 cooperatively dictate cytokine signaling during death receptor activation and indicate that regulated metalloproteinase activity integrates survival and death signals during acute hepatotoxic stress., Introduction Hepatocytes are highly sensitive to death receptor-mediated apoptosis. Engagement of this pathway elicits multiple and complex extrinsic signals, some apoptotic and others hepatoprotective. The cumulative tissue response to these [...]

Loss of Hippo signaling in Drosophila leads to tissue overgrowth as a result of increased cell proliferation and decreased cell death. YAP (a homolog of Drosophila Yorkie and target of the Hippo pathway) was recently implicated in control of organ size, epithelial tissue development, and tumorigenesis in mammals. However, the role of the mammalian Hippo pathway in such regulation has remained unclear. We now show that mice with liver-specific ablation of WW45 (a homolog of Drosophila Salvador and adaptor for the Hippo kinase) manifest increased liver size and expansion of hepatic progenitor cells (oval cells) and eventually develop hepatomas. Moreover, ablation of WW45 increased the abundance of YAP and induced its localization to the nucleus in oval cells, likely accounting for their increased proliferative capacity, but not in hepatocytes. Liver tumors that developed in mice heterozygous for WW45 deletion or with liver-specific WW45 ablation showed a mixed pathology combining characteristics of hepatocellular carcinoma and cholangiocarcinoma and seemed to originate from oval cells. Together, our results suggest that the mammalian Hippo--Salvador pathway restricts the proliferation of hepatic oval cells and thereby controls liver size and prevents the development of oval cell--derived tumors. conditional knockout mice | DDC diet | hepatoma | WW45 | YAP doi/ 10.1073/pnas.0912203107

Subramanian VS, Marchant JS, Said HM. Molecular determinants dictating cell surface expression of the human sodium-dependent vitamin C transporter-2 in human liver cells. Am J Physiol Gastrointest Liver Physiol 298: G267-G274, 2010. First published November 19, 2009; doi: 10.1152/ajpgi.00435.2009.--The human sodium-dependent vitamin C transporter-2 (hSVCT2) plays an important role in cellular accumulation of ascorbic acid in liver cells. However, little is known about the molecular determinants that direct hSVCT2 to the cell surface in hepatocytes. We addressed this issue using live cell imaging methods to resolve the distribution and trafficking of truncated or mutated hSVCT2 constructs in a cellular model of human hepatocytes, HepG2 cells. Whereas a full-length hSVCT2-yellow fluorescent protein (YFP) fusion protein was functionally expressed at the cell surface in HepG2 cells, serial truncation and mutation analysis demonstrated an essential role for both [NH.sub.2]and COOH-terminal sequence(s) for cell surface expression and function. Video-rate confocal imaging showed evidence of dynamic hSVCT2-YFP containing intracellular trafficking vesicles, the motility of which was impaired following disruption of microtubules using nocodazole. However, in a HepG2 cell line stably expressing hSVCT2-YFP at the cell surface, plasma membrane levels of hSVCT2 were unaffected by inhibition of microtubule-associated motor proteins; rather, surface expression of hSVCT2-YFP was increased following treatment with myosin inhibitors. Together, these results show that 1) both [NH.sub.2]- and COOH-terminal sequences are essential for proper localization of hSVCT2, 2) cell surface delivery is dependent on intact microtubules, and 3) peripheral microfilaments regulate insertion and retrieval of hSVCT2 into the plasma membrane. ascorbic acid; uptake; trafficking doi: 10.1152/ajpgi.00435.2009

In mammals, the circadian oscillator generates approximately 24-h rhythms in feeding behavior, even under constant environmental conditions. Livers of mice held under constant darkness exhibit circadian rhythm in abundance in up to 15% of expressed transcripts. Therefore, oscillations in hepatic transcripts could be driven by rhythmic food intake or sustained by the hepatic circadian oscillator, or a combination of both. To address this question, we used distinct feeding and fasting paradigms on wild-type (WT) and circadian clock-deficient mice. We monitored temporal patterns of feeding and hepatic transcription. Both food availability and the temporal pattern of feeding determined the repertoire, phase, and amplitude of the circadian transcriptome in WT liver. In the absence of feeding, only a small subset of transcripts continued to express circadian patterns. Conversely, temporally restricted feeding restored rhythmic transcription of hundreds of genes in oscillator-deficient mouse liver. Our findings show that both temporal pattern of food intake and the circadian clock drive rhythmic transcription, thereby highlighting temporal regulation of hepatic transcription as an emergent property of the circadian system. CREB | metabolism | mouse liver | circadian rhythms doi/ 10.1073/pnas.0909591106

Kren BT, Wong PY-P, Shiota A, Zhang X, Zeng Y, Steer CJ. Polysome trafficking of transcripts and microRNAs in regenerating liver after partial hepatectomy. Am J Physiol Gastrointest Liver Physiol 297: G1181-G1192, 2009. First published October l, 2009; doi: 10.1152/ajpgi.90636.2008.--Liver regeneration after 70% partial hepatectomy (PH) in rats induces >95% of hepatocytes to undergo two rounds of semisynchronous cell replication. Gene expression is controlled primarily by posttranscriptional processing, including changes in mRNA stability. However, the translational activity of a specific mRNA can also be modulated after PH, resulting in significant uncoupling of protein and transcript levels relative to quiescent liver for many genes including c-myc and p53. Although the precise mechanism by which this uncoupling occurs is unknown, the polysoreal association of mRNA and microRNA (miRNA) can significantly modulate rate of decay as well as translational activity. Thus we characterized the association of c-myc and p53 mRNAs and miRNAs in free and cytoskeleton- and membrane-bound polysome populations 3, 6, and 24 h after PH. The transcripts for c-myc and p53 were differentially distributed in the three discrete polysome populations, and this was dramatically modulated during liver regeneration. Nascent polysome-associated p53 and c-myc proteins were also differentially expressed in the free and cytoskeleton- and membrane-bound polysomes and significantly uncoupled from transcript levels relative to nonresected liver. At least 85 miRNAs were associated with the three polysome populations, and their abundance and distribution changed significantly during liver regeneration. These data suggest that posttranscriptional control of c-myc and p53 protein expression is associated with the translocation of transcripts between the different polyribosomes. The alteration of expression for the same transcript in different polysome populations may, in part, be due to the action of miRNAs. c-myc; liver regeneration; p53 doi: 10.1152/ajpgi.90636.2008

Criddle DN, Booth DM, Mukherjee R, McLaughlin E, Green GM, Sutton R, Petersen OH, Reeve JR Jr. Cholecystokinin-58 and cholecystokinin-8 exhibit similar actions on calcium signaling, zymogen secretion, and cell fate in murine pancreatic acinar cells. Am J Physiol Gastrointest Liver Physiol 297: G1085-G1092, 2009. First published October 8, 2009; doi: 10.1152/ajpgi.00119.2009.--The gastrointestinal hormone CCK exists in various molecular forms, with differences in bioactivity between the well-characterized CCK-8 and larger CCK-58 previously reported. We have compared the effects of these peptides on cytosolic calcium concentration ([[[Ca.sup.2+]].sub.c]), mitochondrial metabolism, enzyme secretion, and cell fate in murine isolated pancreatic acinar cells using fluorescence confocal microscopy and patch-clamp electrophysiology. CCK-58 (1-10 pM) induced transient, oscillatory increases of [[[Ca.sup.2+]].sub.c], which showed apical to basolateral progression and were associated with a rise of mitochondrial NAD(P)H. CCK-58 (10 pM) induced zymogen exocytosis in isolated cells and amylase secretion from isolated cells and whole tissues. Hyperstimulation with supraphysiological CCK-58 (5 nM) induced a single large increase of [[[Ca.sup.2+]].sub.c] that declined to a plateau, which remained above the basal level 20 min after application and was dependent on external [[Ca.sup.2+]] entry. In cells dispersed from the same tissues, CCK-8 induced similar patterns of responses to those of CCK-58, with oscillatory increases of [[[Ca.sup.2+]].sub.c] at lower (pM) concentrations and sustained responses at 5 nM. CCK-58 and CCK-8 exhibited similar profiles of action on cell death, with increases in necrosis at high CCK-58 and CCK-8 (10 nM) that were not significantly different between peptides. The present experiments indicate that CCK-8 and CCK-58 have essentially identical actions on the acinar cell at high and low agonist concentrations, suggesting an action via the same receptor and that the differences observed in an intact rat model may result from indirect effects of the peptides. Our data strengthen the argument that CCK-58 is an important physiological form of this gastrointestinal hormone. mitochondria; necrosis; apoptosis doi: 10.1152/ajpgi.00119.2009

Choi SS, Omenetti A, Witek RP, Moylan CA, Syn W, Jung Y, Yang L, Sudan DL, Sicklick JK, Michelotti GA, Rojkind M, Diehl AM. Hedgehog pathway activation and epithelial-to-mesenchymal transitions during myofibroblastic transformation of rat hepatic cells in culture and cirrhosis. Am J Physiol Gastrointest Liver Physiol 297:G1093-G1106, 2009. First published October 8, 2009; doi: 10.1152/ajpgi.00292.2009.--Myofibroblastic hepatic stellate cells (MF-HSC) are derived from quiescent hepatic stellate cells (Q-HSC). Q-HSC express certain epithelial cell markers and have been reported to form junctional complexes similar to epithelial cells. We have shown that Hedgehog (Hh) signaling plays a key role in HSC growth. Because Hh ligands regulate epithelial-to-mesenchymal transition (EMT), we determined whether Q-HSC express EMT markers and then assessed whether these markers change as Q-HSC transition into MF-HSC and whether the process is modulated by Hh signaling. Q-HSC were isolated from healthy livers and cultured to promote myofibroblastic transition. Changes in mRNA and protein expression of epithelial and mesenchymal markers, Hh ligands, and target genes were monitored in HSC treated with and without cyclopamine (an Hh inhibitor). Studies were repeated in primary human HSC and clonally derived HSC from a cirrhotic rat. Q-HSC activation in vitro (culture) and in vivo (C[Cl.sub.4]-induced cirrhosis) resulted in decreased expression of Hh-interacting protein (Hhip, an Hh antagonist), the EMT inhibitors bone morphogenic protein (BMP-7) and inhibitor of differentiation (Id2), the adherens junction component E-cadherin, and epithelial keratins 7 and 19 and increased expression of Gli2 (an Hh target gene) and mesenchymal markers, including the mesenchyme-associated transcription factors Lhx2 and Msx2, the myofibroblast marker [alpha]-smooth muscle actin, and matrix molecules such as collagen. Cyclopamine reverted myofibroblastic transition, reducing mesenchymal gene expression while increasing epithelial markers in rodent and human HSC. We conclude that Hh signaling plays a key role in transition of Q-HSC into MF-HSC. Our findings suggest that Q-HSC are capable of transitioning between epithelial and mesenchymal fates. bone morphogenetic protein-7; cyclopamine; fibrosis; proliferation; regeneration doi: 10.1152/ajpgi.00292.2009

The liver is a major site for the metabolism of xenobiotic compounds due to its abundant level of phase I/II metabolic enzymes. With the cost of drug development escalating to over $400 million/ drug there is an urgent need for the development of rigorous models of hepatic metabolism for preclinical screening of drug clearance and hepatotoxicity. Here, we present a microenvironment in which primary human and rat hepatocytes maintain a high level of metabolic competence without a long adaptation period. We demonstrate that co-cultures of hepatocytes and endothelial cells in serum-free media seeded under 95% oxygen maintain functional apical and basal polarity, high levels of cytochrome P450 activity, and gene expression profiles on par with freshly isolated hepatocytes. These oxygenated co-cultures demonstrate a remarkable ability to predict in vivo drug clearance rates of both rapid and slow clearing drugs with an [R.sup.2] of 0.92. Moreover, as the metabolic function of oxygenated co-cultures stabilizes overnight, preclinical testing can be carried out days or even weeks before other culture methods, significantly reducing associated labor and cost. These results are readily extendable to other culture configurations including three-dimensional culture, bioreactor studies, as well as microfabricated co-cultures. drug discovery | liver metabolism | tissue engineering

The emergence of regenerative medicine has led to significant advances in the identification and understanding of human stem cells and adult progenitor cells. Both cell populations exhibit plasticity and theoretically offer a potential source of somatic cells in large numbers. Such a resource has an important role to play in the understanding of human development, in modeling human disease and drug toxicity, and in the generation of somatic cells in large numbers for cell-based therapies. Presently, liver transplantation is the only effective treatment for end-stage liver disease. Although this procedure can be carried out with high levels of success, the routine transplant of livers is severely limited by organ donor availability. As a result, attention has focused on the ability to restore liver mass and function by alternative approaches ranging from the bioartificial device to transplantation of human hepatocytes. In this review we will focus on the generation of human hepatic endoderm from different stem/progenitor cell populations with a view to its utility in regenerative medicine. embryonic stem cell; function; hepatocyte; induced pluripotent stem cell

Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid--derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response., Introduction In the first phase I/II trial of hepatic gene transfer of adeno-associated virus 2 (AAV2) encoding Factor IX (AAV2-F9) in human hemophilia B subjects, transgene expression was demonstrable but [...]

The concentrative nucleoside transporter-1 (CNT1) is a member of the solute carrier 28 (SLC28) gene family and is expressed in the liver, intestine, and kidneys. CNT1 mediates the uptake of naturally occurring pyrimidine nucleosides, but also nucleoside analogs used in anticancer and antiviral therapy. Thus expression levels of CNT1 may affect the pharmacokinetics of these drugs and the outcome of drug therapy. Because little is known about the transcriptional regulation of human CNT1 gene expression, we have characterized the CNT1 promoter with respect to DNA response elements and their binding factors. The transcriptional start site of the CNT1 gene was determined by 5'-RACE. In silico analysis revealed the existence of three putative binding sites for the nuclear receptor hepatocyte nuclear factor-4[alpha] (HNF-4[alpha]) within the CNT1 promoter. A luciferase reporter gene construct containing the CNT1 promoter region was transactivated by HNF-4[alpha] in human cell lines derived from the liver, intestine, and kidneys. Consistent with this, we showed in electromobility shift assays that HNF-4[alpha] specifically binds to two conserved direct repeat-1 motifs within the proximal CNT1 promoter. In cotransfection experiments, the transcriptional coactivator peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha] further increased, whereas the bile acid-inducible corepressor small heterodimer partner reduced, HNF-4[alpha]-dependent CNT1 promoter activity. Consistent with the latter phenomenon, CNT1 mRNA expression levels were suppressed in primary human hepatocytes upon bile acid treatment. Supporting the physiological relevance and species conservation of this effect, ileal Cnt1 mRNA expression was decreased upon bile acid feeding and increased upon bile duct ligation in mice. nucleoside transport; transcriptional regulation; nuclear receptors

We have investigated the regulation of translation during the period of rapid liver growth that occurs at the end of gestation in the rat. This work was based on our prior observation that fetal hepatocyte proliferation is resistant to the inhibitory effects of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a nutrient-sensing kinase that controls ribosome biogenesis and protein translation. We hypothesized that translation control in late-gestation fetal liver differs from that in adult liver. We first examined the ability of rapamycin to inhibit the translation of mRNAs encoding ribosomal proteins. Consistent with the effect of rapamycin on proliferation, the activation of adult liver 5'-terminal oligopyrimidine tracts (5'-TOP) translation that occurred during refeeding after food deprivation was sensitive to rapamycin. Fetal liver 5'-TOP translation was insensitive. We went on to examine the eukaryotic initiation factor (eIF) 4F cap-binding complex that controls global protein synthesis. The molecular weights of the multiple eIF4G1 isoforms present in fetal and adult liver eIF4F complexes differed. In addition, fetal liver expressed the eIF4A1 form of the eIF4A helicase, whereas adult liver contained eIF4AI and eIF4A2. Rapamycin administration before refeeding in adult rats inhibited formation of the preinitiation complex to a much greater degree than rapamycin administration to fetal rats in situ. We conclude that there are major structural and functional differences in translation control between late-gestation fetal and adult liver. These differences may confer differential sensitivity to the growth inhibitory effects of rapamycin. liver; hepatocyte; mammalian target of rapamycin; signal transduction; ribosome

Reduction of plasma cholesterol by citrus flavonoids is associated with effects on specific liver functions related to lipid handling. In previous in vivo studies, polymethoxylated flavones (PM F) reduced plasma cholesterol levels at lower doses than required for flavanones. To delineate hepatic mechanisms that underlie this differential potency, we used HepG2 cells to quantitate effects on expression of the LDL receptor (LDLR) gene. A dose-response analysis showed that 200 [micro]mol/L hesperetin, a flavanone present as a disaccharide in oranges, increased LDLR mRNA levels 3.6- to 4.7-fold of the untreated control. In contrast, nobiletin, a PM F found at the highest concentration in oranges and tangerines, achieved maximal stimulation of 1.5- to 1.6-fold of control at only 5 [micro]mol/L. Transcriptional regulation of the LDLR gene by citrus flavonoids has been implicated but, to our knowledge, not directly demonstrated. Here, using transfection vector constructs containing the upstream region of the LDLR gene, we show differences in both potency and efficacy in the induction of transcription, with peak stimulation of 5.3- to 7.5-fold of control at 150-160 [micro]mol/L hesperetin and 3- to 3.8-fold of control at 10-20 [micro]mol/L nobiletin. Hesperetin sustains induction, whereas nobiletin is inhibitory at high doses, resulting in an inverted-U dose response. The sterol regulatory element (SRE) in the LDLR gene upstream region plays a crucial role, because mutation of this site strongly attenuated induction in response to hesperetin or nobiletin. Thus, citrus flavonoids are likely to act through the SRE- binding proteins, with PMF initially activating these mechanisms at considerably lower concentrations than flavanones.

Several studies are conducted to study the association between the common polymorphisms of the HNF1A gene encoding hepatocyte nuclear factor-1[alpha] and plasma C-reactive protein (CRP) concentration. The two discussed phenomenon are shown to be completely related and dependent on each other.

In the liver, the JNK cascade is induced downstream of TNF receptors (TNFRs) in response to inflammatory, microbial, and toxic challenges. Sustained activation of JNK triggers programmed cell death (PCD), and hepatocyte survival during these challenges requires induction of the NF-[kappa]B pathway, which antagonizes this activation by upregulating target genes. Thus, modulation of JNK activity is crucial to the liver response to TNFR-mediated challenge. The basis for this modulation, however, is unknown. Here, we investigated the role of the NF-[kappa]B target Gadd45b in the regulation of hepatocyte fate during liver regeneration after partial hepatectomy. We generated Gadd45b-/- mice and found that they exhibited decreased hepatocyte proliferation and increased PCD during liver regeneration. Notably, JNK activity was markedly increased and sustained in livers of Gadd45b-/- mice compared with control animals after partial hepatectomy. Furthermore, imposition of a Jnk2-null mutation, attenuating JNK activity, completely rescued the regenerative response in Gadd45b-/- mice. Interestingly, Gadd45[beta] ablation did not affect hepatotoxic JNK signaling after a TNFR-mediated immune challenge, suggesting specificity in the inducible hepatic program for JNK restraint activated during distinct TNFR-mediated challenges. These data provide a basis for JNK suppression during liver regeneration and identify Gadd45[beta] as a potential therapeutic target in liver diseases., Introduction TNF-[alpha] is a pleiotropic cytokine that plays important roles in immunity, inflammation, and tumorigenesis (1), (2). Exposure to TNF-[alpha] induces either cell proliferation or programmed cell death (PCD) through [...]

Human species C adenovirus serotype 5 (Ad5) is the most common viral vector used in clinical studies worldwide. Ad5 vectors infect liver cells in vivo with high efficiency via a poorly defined mechanism, which involves virus binding to vitamin K-dependent blood coagulation factors. Here, we report that the major Ad5 capsid protein, hexon, binds human coagulation factor X (FX) with an affinity of 229 pM. This affinity is 40-fold stronger than the reported affinity of Ad5 fiber for the cellular receptor coxsackievirus and adenovirus receptor, CAR. Cryoelectron microscopy and single-particle image reconstruction revealed that the FX attachment site is localized to the central depression at the top of the hexon trimer. Hexon-mutated virus bearing a large insertion in hexon showed markedly reduced FX binding in vitro and failed to deliver a transgene to hepatocytes in vivo. This study describes the mechanism of FX binding to Ad5 and demonstrates the critical role of hexon for virus infection of hepatocytes in vivo. gene transfer | virus targeting

Hepatocyte nuclear factor-1[beta] (HNF-1[beta]) is a Pit-1, Oct-1/2, Unc-86 (POU) homeodomain-containing transcription factor expressed in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1[beta] cause maturity-onset diabetes of the young, type 5 (MODY5), which is characterized by early-onset diabetes mellitus and congenital malformations of the kidney, pancreas, and genital tract. Knockout of HNF-1[beta] in the mouse kidney results in cyst formation. However, the signaling pathways and transcriptional programs controlled by HNF-1[beta] are poorly understood. Using genome-wide chromatin immunoprecipitation and DNA microarray (ChiP-chip) and microarray analysis of mRNA expression, we identified SOCS3 (suppressor of cytokine signaling-3) as a previously unrecognized target gene of HNF-1[beta] in the kidney. HNF-1[beta] binds to the SOCS3 promoter and represses SOCS3 transcription. The expression of SOCS3 is increased in HNF-1[beta] knockout mice and in renal epithelial cells expressing dominant-negative mutant HNF-1[beta]. Increased levels of SOCS-3 inhibit HGF-induced tubulogenesis by decreasing phosphorylation of Erk and STAT-3. Conversely, knockdown of SOCS-3 in renal epithelial cells expressing dominant-negative mutant HNF-1[beta] rescues the defect in HGF-induced tubuIogenesis by restoring phosphorylation of Erk and STAT-3. Thus, HNF-1[beta] regulates tubulogenesis by controlling the levels of SOCS-3 expression. Manipulating the levels of SOCS-3 may be a useful therapeutic approach for human diseases induced by HNF-1[beta] mutations. chromatin | kidney | transcription | tubulogenesis | TCF2

Analysis of the molecular factors determining hepatocyte survival or death in response to inflammatory stimuli is essential for understanding the pathogenesis of inflammatory liver disease and for identifying novel therapeutic approaches, c-Jun N-terminal kinase (JNK) is a major mediator of cytokine-induced cell death during hepatitis, but the signaling pathways downstream of JNK remain less well defined. Here we show that the transcription factor c-Jun/AP-1, a prototypic target of JNK, is strongly expressed in the liver of patients with acute liver injury. The molecular function of c-Jun in inflammatory liver disease was analyzed in mice by using the Con A model of T cell-mediated hepatitis. Mice lacking c-Jun in hepatocytes display increased liver cell death and mortality upon Con A injection. This phenotype is caused by impaired expression of inducible nitric oxide synthase (nos2), a direct transcriptional target of c-Jun, and reduced production of hepatoprotective nitric oxide (NO). Moreover, increased hepatotoxicity in mutant mice is likely caused by hypoxia and oxidative stress and can be rescued pharmacologically by liver-specific NO delivery. These findings demonstrate that c-Jun/AP-1 is hepatoprotective during acute hepatitis by regulating nos2/NO expression and thus functionally antagonizes the cell death-promoting functions of JNK. activator protein 1 | concanavalin A | hypoxia

Cholestatic disorders may arise from liver ischemia (e.g., in liver transplantation) through various mechanisms. We have examined the potential of hypoxia to induce changes in the expression of hepatobiliary transporter genes. In a model of arterial liver ischemia subsequent to complete arterial deprivation of the rat liver, the mRNA levels of VEGF, a hypoxia-inducible gene, were increased fivefold after 24 h. The pattern of VEGF-induced expression and ultrastructural changes, including swelling of the endoplasmic reticulum, indicated that hypoxia affected primarily cholangiocytes, but also hepatocytes, predominantly in the periportal area. Serum and bile analyses demonstrated liver dysfunction of cholestatic type with reduced bile acid biliary excretion. Fluorescence-labeled ursodeoxycholic acid used as a tracer displayed no regurgitation, eliminating bile leakage as a significant mechanism of cholestasis in this model. In liver tissue, a marked reduction in the mRNA levels of [Na.sup.+]-taurocholate-cotransporting polypeptide (Ntcp), bile salt export protein (Bsep), and multidrug resistance-associated protein 2 (Mrp2) and an increase in those of Cftr were detected before bile duct proliferation occurred. In cultured hepatocytes, a nontoxic hypoxic treatment caused a decrease in the mRNA and protein expression of Ntcp, Bsep, and Mrp2 and in the mRNA levels of nuclear factors involved in the transactivation of these genes, i.e., HNF4[alpha], RXR[alpha], and FXR. In bile duct preparations, hypoxic treatment elicited an increase in Cftr transcripts, along with a rise in cAMP, a major regulator of Cftr expression and function. In conclusion, hypoxia triggers a downregulation of hepatocellular transporters, which may contribute to cholestasis, whereas Cftr, which drives secretion in cholangiocytes, is upregulated. liver ischemia; cholestasis; [Na.sup.+]-taurocholate-cotransporting polypeptide; bile salt export pump; multidrug resistance-associated protein 2; cystic fibrosis transmembrane conductance regulator doi:10.1152/ajpgi.00175.2006

BACKGROUND: Toxicogenomics experiments often reveal thousands of transcript alterations that are related to multiple processes, making it difficult to identify key gene changes that are related to the toxicity of [...]

Fatty acids and glucose are strong modulators of the expression of glucose-6-phosphatase (GIc-6-Pase), an enzyme that plays a key role in glucose homeostasis. PUFA inhibit, whereas SFA and monounsaturated fatty acids induce the expression of the GIc-6-Pase gene. Palmitate and oleate are the most abundant fatty acid species in circulation during food deprivation in mammals. Although dietary fats have been shown to modulate the expression of genes involved in both lipid and carbohydrate metabolism in liver, little is known regarding the molecular mechanism of transcriptional response of the GIc-6-Pase gene to long-chain fatty acids. Using H411E hepatoma cells and hepatocytes from adult rats, we investigated the mechanism of the induction of this gene by palmitate and oleate. Both of these fatty acids stimulated GIc-6-Pase gene transcription but did not affect the stability of its mRNA. In transient transfection assays, transcription from the GIc-6-Pase gene promoter was markedly enhanced by both palmitate and oleate but not by arachidonate. Chromatin immunoprecipitation analysis was used to show that palmitate induced the recruitment of an array of transcription factors viz hepatic nuclear factor(NF)-4[alpha], CAAT/enhancer binding protein[beta], PPAR[alpha], chicken ovalbumin upstream promoter transcription factor (COUP-TF), cAMP regulatory element binding protein, and NF-[kappa]B to this gene promoter. Although it is presently unclear how these various transcription factors interact at this promoter, the data are consistent with the view that multiple regulatory elements in the GIc-6-Pase gene promoter are responsible for the modulation of gene transcription by fatty acids.

We examined the role of hepatic heparan sulfate in triglyceride-rich lipoprotein metabolism by inactivating the biosynthetic gene GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) in hepatocytes using the Cre-loxP system, which resulted in [...]

EPh kinases and their ephrin ligands are widely expressed in epithelial cells in vitro and in vivo. Our results show that activation of endogenous EphA kinases in Madin-Darby canine kidney (MDCK) cells negatively regulates hepatocyte growth factor/scatter factor (HGF)-induced branching morphogenesis in collagen gel. Cotreatment with HGF and ephrin-A1 reduced sprouting of cell protrusions, an early step in branching morphogenesis. Moreover, addition of ephrin-A1 after HGF stimulation resulted in collapse and retraction of preexisting cell protrusions. In a newly developed assay that simulates the localized interactions between Ephs and ephrins in vivo, immobilized ephrin-A1 suppressed HGF-induced MDCK cell scattering. Ephrin-A1 inhibited basal ERK1/2 mitogen-activated protein kinase activity; however, the ephrin-A1 effect on cell protrusion was independent of the mitogen-activated protein kinase pathway. Ephrin-A1 suppressed HGF-induced activation of Rac1 and p21-activated kinase, whereas RhoA activation was retained, leading to the preservation of stress fibers. Moreover, dominant-negative RhoA or inhibitor of Rho-associated kinase (Y27632) substantially negated the inhibitory effects of ephrin-A1. These data suggest that interfering with c-Met signaling to Rho GTPases represents a major mechanism by which EphA kinase activation inhibits HGF-induced MDCK branching morphogenesis.

Recent work shows that S-adenosylmethionine (AdoMet) helps maintain normal liver function as chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. The mechanisms by which these nontraditional functions of AdoMet occur are unknown. Here, we use knockout mice deficient in hepatic AdoMet synthesis ([MAT1A.sup.-/-]) to study the proteome of the liver during the development of steatohepatitis. One hundred and seventeen protein spots, differentially expressed during the development of steatohepatitis, were selected and identified by peptide mass fingerprinting. Among them, 12 proteins were found to be affected from birth, when [MAT1A.sup.-/-] expression is switched on in WT mouse liver, to the rise of histological lesions, which occurs at [approximately equal to] 8 months. Of the 12 proteins, 4 [prohibitin 1 (PHB1), cytochrome c oxidase I and II, and ATPase [beta]-subunit] have known roles in mitochondrial function. We show that the alteration in expression of PHB1 correlates with a loss of mitochondrial function. Experiments in isolated rat hepatocytes indicate that AdoMet regulates PHB1 content, thus suggesting ways by which steatohepatitis may be induced. Importantly, we found the expression of these mitochondrial proteins was abnormal in ob/ob mice and obese patients who are at risk for nonalcoholic steatohepatitis.

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA-binding domain and whose members play essential roles in cellular proliferation, differentiation, transformation, longevity, and metabolic homeostasis. Liver regeneration studies with transgenic mice demonstrated that FoxM1B regulates the onset of hepatocyte DNA replication and mitosis by stimulating expression of cell cycle genes. Here, we demonstrate that albumin-promoter-driven Cre recombinase-mediated hepatocyte-specific deletion of the Foxm1b Floxed (fl) targeted allele resulted in significant reduction in hepatocyte DNA replication and inhibition of mitosis after partial hepatectomy. Reduced DNA replication in regenerating [Foxm1b.sup.-/-] hepatocytes was associated with sustained increase in nuclear staining of the cyclin-dependent kinase (Cdk) inhibitor [p21.sup.Cip1] (p21) protein between 24 and 40 h after partial hepatectomy. Furthermore, increased nuclear p21 levels and reduced expression of Cdc25A phosphatase coincided with decreases in Cdk2 activation and hepatocyte progression into S-phase. Moreover, the significant reduction in hepatocyte mitosis was associated with diminished mRNA levels and nuclear expression of Cdc25B phosphatase and delayed accumulation of cyclin B1 protein, which is required for Cdk1 activation and entry into mitosis. Cotransfection studies demonstrate that FoxM1B protein directly activated transcription of the Cdc25B promoter region. Our present study shows that the mammalian Foxm1b transcription factor regulates expression of cell cycle proteins essential for hepatocyte entry into DNA replication and mitosis. knock-out mouse | Cdc25A | Cdc25B | cyclin-dependent kinase inhibitor [p21.sup.Cip1]

Research has been conducted on the tumor metastasis-associated proteins. Results demonstrate that these proteins cooperate and the role of ERM proteins and their link to the cortical actin cytoskeleton in signal transfer have been investigated.

Cerebellar development occurs mainly postnatally and implies cell proliferation and migration. Hepatocyte growth factor (HGF) and Met are involved in mediating these responses in other tissues and are coexpressed in the cerebellum. Here we show that Met is localized in granule cell precursors and that cultures of these cells respond to HGF with proliferation. To study the role of HGF and Met in the cerebellum in vivo, we produced a viable hypomorphic Met mutant by knocking in the met locus a point mutation to abrogate the receptor Grb2-binding site. A similar mutant was previously described as perinatal lethal. In this 'first-generation' knock-in the recombinant locus retained the Neo cassette ([Met.sup.grb2/grb2neo+]). In the knock-in presented here Neo was Loxed and excised by Cre recombinase, which led to higher tissue levels of Metgrb2 protein, sufficient to rescue viability. In [Met.sup.grb2/grb2neo-] mice the size of the cerebellum was reduced and foliation defects were evident, especially in the central and posterior half of the vermis. Proliferation of granule precursors in vivo was 25% lower than in controls. In cultures of mutant granule cells HGF-induced microtubule-associated protein kinase activation was reduced and transient. Behavioral tests indicated a balance impairment in [Me.sup.tgrb2/grb2neo-] mice. Altogether these data indicate that normal cerebellar development and, possibly, function, require HGF and Met, and that proliferation of granule cells in the cerebellum critically depends on full HGF/Met signaling.

Mice lacking the stress-signaling kinase SEK1 die from embryonic day 10.5 (E10.5) to E12.5. Although a defect in liver formation is accompanied with the embryonic lethality of sek[1.sup.-/-] mice, the mechanism of the liver defect has remained unknown. In the present study, we first produced a monoclonal antibody specifically recognizing murine hepatoblasts for the analysis of liver development and further investigated genetic interaction of sek1 with tumor necrosis factor-[alpha] receptor 1 gene (tnfr1) and protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek[1.sup.-/-] embryos was not protected by additional tnfr1 mutation, which rescues the embryonic lethality of mice lacking NF-[kappa]B signaling components. There was a progressive increase in the hepatoblast cell numbers of wild-type embryos from E10.5 to E12.5. Instead, impaired hepatoblast proliferation was observed in sek[1.sup.-/-] livers from E10.5, though fetal liver-specific gene expression was normal. The impaired phenotype in sek[1.sup.-/-] livers was more severe than in [c-jun.sup.-/-] embryos, and sek[1.sup.-/-] [c-jun.sup.-/-] embryos died more rapidly before E8.5. The hepatoblast proliferation required no hematopoiesis, since liver development was not impaired in AML[1.sup.-/-] mice that lack hematopoietic functions. Stimulation of stress-activated protein kinase/c-Jun N-terminal kinase by hepatocyte growth factor was attenuated in sek[1.sup.-/-] livers. Thus, SEK1 appears to play a crucial role in hepatoblast proliferation and survival in a manner apparently different from NF-[kappa]B or c-Jun. Key words: SEK1; NF-[kappa]B; c-Jun; HGF; hematopoiesis; hepatogenesis.

Research has been carried out on the hepatocyte nuclear factor 3(HNF-3)/fork head family. The structural study of the DNA binding domain of the conserved winged helix protein HNF-1 and its DNA components has been carried out and the results are discussed.

Tissue-specific deletion of Foxa2 in pancreatic beta cells has been found to result in hyperinsulinemic hypoglycemia. Mice having no Foxa2 specifically in pancreatic beta cells were generated and its contribution to beta-cell development and physiology analyzed.

A very powerful procedure was used to find what happens when HNF-4alpha function is absent in liver development, and it was found that mammalian hepatocyte differentiation must have the this transcription factor. Mouse embryos die if they have no HNG-4alpha, but they were rescued by complementation with a tetraploid embryo-derived wild-type visceral endoderm.

A high protein concentration in the diet induces the gene expression of several amino acid degrading enzymes such as histidase (Hal) in rats. It is important to understand whether the amino acid pattern of the dietary protein affects the gene expression of these enzymes. The purpose of the present work was to study the effect of a histidine-imbalanced diet on the activity and mRNA concentration of rat hepatic histidase. Seven groups of six rats were fed one of the following diets: 1) 6% casein (basal), 2) 20% casein, 3) 35% casein, 4) an imbalance diet containing 6% casein plus a mixture of indispensable amino acids (IAA) equivalent to a 20% casein diet without histidine (1-20), 5) 6% casein plus a mixture of IAA equivalent to a 35% casein diet without histidine (I-35), 6) a corrected diet containing 6% casein plus IAA including histidine equivalent to a 20% casein diet, 7) a corrected diet containing 6% casein plus IAA including histidine equivalent to a 35% casein diet. Serum histidine concentration was inversely proportional to the protein content of the diet, and it was significantly higher in rats fed the corrected diets compared to their respective imbalanced diet groups. Hal activity increased as the protein content of the diet increased. Greater histidine imbalance resulted in lower food intake and higher Hal activity. Rats fed histidine-corrected diets had lower activity than their respective imbalanced groups. Differences in Hal activity were associated with differences in the concentration of Hal mRNA. These results indicate that rats fed a histidine-imbalanced diet exhibit reduced food intake and weight gain and increased Hal gene expression as a consequence of an increased amino acid catabolism. KEY WORDS: amino acid imbalance; gene expression; histidase; histidine; rats

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism. Affected individuals evidence iron accumulation in tissue parenchyma in association with absent serum ceruloplasmin. Genetic studies of such patients reveal inherited mutations in the ceruloplasmin gene. To elucidate the role of ceruloplasmin in iron homeostasis, we created an animal model of aceruloplasminemia by disrupting the murine ceruloplasmin (Cp) gene. Although normal at birth, [Cp.sup.-/-] mice demonstrate progressive accumulation of iron such that by one year of age all animals have a prominent elevation in serum ferritin and a 3- to 6-fold increase in the iron content of the liver and spleen. Histological analysis of affected tissues in these mice shows abundant iron stores within reticuloendothelial cells and hepatocytes. Ferrokinetic studies in [Cp.sup.+/+] and [Cp.sup.-/-] mice reveal equivalent rates of iron absorption and plasma iron turnover, suggesting that iron accumulation results from altered compartmentalization within the iron cycle. Consistent with this concept, [Cp.sup.-/-] mice showed no abnormalities in cellular iron uptake but a striking impairment in the movement of iron out of reticuloendothelial cells and hepatocytes. Our findings reveal an essential physiologic role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores.