Aldh1l2 knockout mouse metabolomics links the loss of the mitochondrial folate enzyme to deregulation of a lipid metabolism observed in rare human disorder.

BACKGROUND: Mitochondrial folate enzyme ALDH1L2 (aldehyde dehydrogenase 1 family member L2) converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2 simultaneously producing NADPH. We have recently reported that the lack of the enzyme due to compound heterozygous mutations was associated with neuro-ichthyotic syndrome in a male patient. Here, we address the role of ALDH1L2 in cellular metabolism and highlight the mechanism by which the enzyme regulates lipid oxidation. METHODS: We generated Aldh1l2 knockout (KO) mouse model, characterized its phenotype, tissue histology, and levels of reduced folate pools and applied untargeted metabolomics to determine metabolic changes in the liver, pancreas, and plasma caused by the enzyme loss. We have also used NanoString Mouse Inflammation V2 Code Set to analyze inflammatory gene expression and evaluate the role of ALDH1L2 in the regulation of inflammatory pathways. RESULTS: Both male and female Aldh1l2 KO mice were viable and did not show an apparent phenotype. However, H&E and Oil Red O staining revealed the accumulation of lipid vesicles localized between the central veins and portal triads in the liver of Aldh1l2-/- male mice indicating abnormal lipid metabolism. The metabolomic analysis showed vastly changed metabotypes in the liver and plasma in these mice suggesting channeling of fatty acids away from ß-oxidation. Specifically, drastically increased plasma acylcarnitine and acylglycine conjugates were indicative of impaired ß-oxidation in the liver. Our metabolomics data further showed that mechanistically, the regulation of lipid metabolism by ALDH1L2 is linked to coenzyme A biosynthesis through the following steps. ALDH1L2 enables sufficient NADPH production in mitochondria to maintain high levels of glutathione, which in turn is required to support high levels of cysteine, the coenzyme A precursor. As the final outcome, the deregulation of lipid metabolism due to ALDH1L2 loss led to decreased ATP levels in mitochondria. CONCLUSIONS: The ALDH1L2 function is important for CoA-dependent pathways including ß-oxidation, TCA cycle, and bile acid biosynthesis. The role of ALDH1L2 in the lipid metabolism explains why the loss of this enzyme is associated with neuro-cutaneous diseases. On a broader scale, our study links folate metabolism to the regulation of lipid homeostasis and the energy balance in the cell.

¹³C magnetic resonance spectroscopy detection of changes in serine isotopomers reflects changes in mitochondrial redox status.

The glycine cleavage system (GCS), the major pathway of glycine catabolism in liver, is found only in the mitochondria matrix and is regulated by the oxidized nicotinamide adenine dinucleotide (NAD(+) )/reduced nicotinamide adenine dinucleotide (NADH) ratio. In conjunction with serine hydroxymethyltransferase, glycine forms the 1 and 2 positions of serine, while the 3 position is formed exclusively by GCS. Therefore, we sought to exploit this pathway to show that quantitative measurements of serine isotopomers in liver can be used to monitor the NAD(+) /NADH ratio using (13) C NMR spectroscopy. Rat hepatocytes were treated with modulators of GCS activity followed by addition of 2-(13) C-glycine, and the changes in the proportions of newly synthesized serine isotopomers were compared to controls. Cysteamine, a competitive inhibitor of GCS, prevented formation of mitochondrial 3-(13) C-serine and 2,3-(13) C-serine isotopomers while reducing 2-(13) C-serine by 55%, demonstrating that ca. 20% of glycine-derived serine is produced in the cytosol. Glucagon, which activates GCS activity, and the mitochondrial uncoupler carbonyl cyanide-3-chlorophenylhydrazone both increased serine isotopomers, whereas rotenone, an inhibitor of complex I, had the opposite effect. These results demonstrate that (13) C magnetic resonance spectroscopy monitoring of the formation of serine isotopomers in isolated rat hepatocytes given 2-(13) C-glycine reflects the changes of mitochondrial redox status.

Inhibition of the mitochondrial permeability transition by protein kinase A in rat liver mitochondria and hepatocytes.

NO and cGMP administered at reperfusion after ischaemia prevent injury to hepatocytes mediated by the MPT (mitochondrial permeability transition). To characterize further the mechanism of protection, the ability of hepatic cytosol in combination with cyclic nucleotides to delay onset of the calcium-induced MPT was evaluated in isolated rat liver mitochondria. Liver cytosol plus cGMP or cAMP dose-dependently inhibited the MPT, required ATP hydrolysis for inhibition and did not inhibit mitochondrial calcium uptake. Specific peptide inhibitors for PKA (protein kinase A), but not PKG (protein kinase G), abolished cytosol-induced inhibition of MPT onset. Activity assays showed a cGMP- and cAMP-stimulated protein kinase activity in liver cytosol that was completely inhibited by PKI, a PKA peptide inhibitor. Size-exclusion chromatography of liver cytosol produced a single peak of cGMP/cAMP-stimulated kinase activity with an estimated protein size of 180-220 kDa. This fraction was PKI-sensitive and delayed onset of the MPT. Incubation of active catalytic PKA subunit directly with mitochondria in the absence of cytosol and cyclic nucleotide also delayed MPT onset, and incubation with purified outer membranes led to phosphorylation of a major 31 kDa band. After ischaemia, administration at reperfusion of membrane-permeant cAMPs and cAMP-mobilizing glucagon prevented reperfusion injury to hepatocytes. In conclusion, PKA in liver cytosol activated by cGMP or cAMP acts directly on mitochondria to delay onset of the MPT and protect hepatocytes from cell death after ischaemia/reperfusion.

Knockout of Putative Tumor Suppressor Aldh1l1 in Mice Reprograms Metabolism to Accelerate Growth of Tumors in a Diethylnitrosamine (DEN) Model of Liver Carcinogenesis.

Cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) is commonly downregulated in human cancers through promoter methylation. We proposed that ALDH1L1 loss promotes malignant tumor growth. Here, we investigated the effect of the Aldh1l1 mouse knockout (Aldh1l1-/-) on hepatocellular carcinoma using a chemical carcinogenesis model. Fifteen-day-old male Aldh1l1 knockout mice and their wild-type littermate controls (Aldh1l1+/+) were injected intraperitoneally with 20 µg/g body weight of DEN (diethylnitrosamine). Mice were sacrificed 10, 20, 28, and 36 weeks post-DEN injection, and livers were examined for tumor multiplicity and size. We observed that while tumor multiplicity did not differ between Aldh1l1-/- and Aldh1l1+/+ animals, larger tumors grew in Aldh1l1-/- compared to Aldh1l1+/+ mice at 28 and 36 weeks. Profound differences between Aldh1l1-/- and Aldh1l1+/+ mice in the expression of inflammation-related genes were seen at 10 and 20 weeks. Of note, large tumors from wild-type mice showed a strong decrease of ALDH1L1 protein at 36 weeks. Metabolomic analysis of liver tissues at 20 weeks showed stronger differences in Aldh1l1+/+ versus Aldh1l1-/- metabotypes than at 10 weeks, which underscores metabolic pathways that respond to DEN in an ALDH1L1-dependent manner. Our study indicates that Aldh1l1 knockout promoted liver tumor growth without affecting tumor initiation or multiplicity.

Closure of VDAC causes oxidative stress and accelerates the Ca(2+)-induced mitochondrial permeability transition in rat liver mitochondria.

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca(2+)-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O(2)(-)) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O(2)(-) from the IMS and sensitizing to the Ca(2+)-induced MPT. Treatment of isolated rat liver mitochondria with 5microM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8+/-1.4min within a range of 100-250microM Ca(2+). G3139-mediated acceleration of the MPT was reversed by 20microM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O(2)(-) in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O(2)(-) accumulation. Mathematical modeling of generation and turnover of O(2)(-) within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O(2)(-). In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O(2)(-), which causes an internal oxidative stress and sensitizes mitochondria toward the Ca(2+)-induced MPT.

Minocycline and N-methyl-4-isoleucine cyclosporin (NIM811) mitigate storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial permeability transition.

UNLABELLED: Graft failure after liver transplantation may involve mitochondrial dysfunction. We examined whether prevention of mitochondrial injury would improve graft function. Orthotopic rat liver transplantation was performed after 18 hours' cold storage in University of Wisconsin solution and treatment with vehicle, minocycline, tetracycline, or N-methyl-4-isoleucine cyclosporin (NIM811) of explants and recipients. Serum alanine aminotransferase (ALT), necrosis, and apoptosis were assessed 6 hours after implantation. Mitochondrial polarization and cell viability were assessed by intravital microscopy. Respiration and the mitochondrial permeability transition (MPT) were assessed in isolated rat liver mitochondria. After transplantation with vehicle or tetracycline, ALT increased to 5242 U/L and 4373 U/L, respectively. Minocycline and NIM811 treatment decreased ALT to 2374 U/L and 2159 U/L, respectively (P < 0.01). Necrosis and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) also decreased from 21.4% and 21 cells/field, respectively, after vehicle to 10.1% and 6 cells/field after minocycline and to 8.7% and 5.2 cells/field after NIM811 (P < 0.05). Additionally, minocycline decreased caspase-3 activity in graft homogenates (P < 0.05). Long-term graft survival was 27% and 33%, respectively, after vehicle and tetracycline treatment, which increased to 60% and 70% after minocycline and NIM811 (P < 0.05). In isolated mitochondria, minocycline and NIM811 but not tetracycline blocked the MPT. Minocycline blocked the MPT by decreasing mitochondrial Ca(2+) uptake, whereas NIM811 blocks by interaction with cyclophilin D. Intravital microscopy showed that minocycline and NIM811 preserved mitochondrial polarization and cell viability after transplantation (P < 0.05). CONCLUSION: Minocycline and NIM811 attenuated graft injury after rat liver transplantation and improved graft survival. Minocycline and/or NIM811 might be useful clinically in hepatic surgery and transplantation.

Cytosolic 10-formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver.

ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1-/- mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1-/- mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1-/- mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1-/- mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.

Deleterious mutations in ALDH1L2 suggest a novel cause for neuro-ichthyotic syndrome.

Neuro-ichthyotic syndromes are a group of rare genetic diseases mainly associated with perturbations in lipid metabolism, intracellular vesicle trafficking, or glycoprotein synthesis. Here, we report a patient with a neuro-ichthyotic syndrome associated with deleterious mutations in the ALDH1L2 (aldehyde dehydrogenase 1 family member L2) gene encoding for mitochondrial 10-formyltetrahydrofolate dehydrogenase. Using fibroblast culture established from the ALDH1L2-deficient patient, we demonstrated that the enzyme loss impaired mitochondrial function affecting both mitochondrial morphology and the pool of metabolites relevant to ß-oxidation of fatty acids. Cells lacking the enzyme had distorted mitochondria, accumulated acylcarnitine derivatives and Krebs cycle intermediates, and had lower ATP and increased ADP/AMP indicative of a low energy index. Re-expression of functional ALDH1L2 enzyme in deficient cells restored the mitochondrial morphology and the metabolic profile of fibroblasts from healthy individuals. Our study underscores the role of ALDH1L2 in the maintenance of mitochondrial integrity and energy balance of the cell, and suggests the loss of the enzyme as the cause of neuro-cutaneous disease.

CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase.

ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the ALDH1L1 gene is commonly silenced through the promoter methylation. It was suggested that ALDH1L1 limits proliferation capacity of the cell and thus functions as putative tumor suppressor. In contrast to cancer cells, mouse cell lines NIH3T3 and AML12 do express the ALDH1L1 protein. In the present study, we show that the levels of ALDH1L1 in these cell lines fluctuate throughout the cell cycle. During S-phase, ALDH1L1 is markedly down regulated at the protein level. As the cell cultures become confluent and cells experience increased contact inhibition, ALDH1L1 accumulates in the cells. In agreement with this finding, NIH3T3 cells arrested in G1/S-phase by a thymidine block completely lose the ALDH1L1 protein. Treatment with the proteasome inhibitor MG-132 prevents such loss in proliferating NIH3T3 cells, suggesting the proteasomal degradation of the ALDH1L1 protein. The co-localization of ALDH1L1 with proteasomes, demonstrated by confocal microscopy, supports this mechanism. We further show that ALDH1L1 interacts with the chaperone-dependent E3 ligase CHIP, which plays a key role in the ALDH1L1 ubiquitination and degradation. In NIH3T3 cells, silencing of CHIP by siRNA halts, while transient expression of CHIP promotes, the ALDH1L1 loss. The downregulation of ALDH1L1 is associated with the accumulation of the ALDH1L1 substrate 10-formyltetrahydrofolate, which is required for de novo purine biosynthesis, a key pathway activated in S-phase. Overall, our data indicate that CHIP-mediated proteasomal degradation of ALDH1L1 facilitates cellular proliferation.

Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after Met-beta-catenin dissociation in hepatocytes.

Hepatocyte growth factor (HGF) and Wnt signaling pathways have been shown to be important in embryogenesis and carcinogenesis. The aim of this study was to elucidate the mechanism of functional similarities observed in the two pathways. We used normal rat liver, primary hepatocyte cultures and a dominant-negative Met expression system to study the effect of HGF on Wnt pathway components. We demonstrate novel association of beta-catenin and Met, a tyrosine kinase receptor of HGF, at the inner surface of the hepatocyte membrane. HGF induces dose-dependent nuclear translocation of beta-catenin in primary hepatocyte cultures that is Wnt independent. The source of beta-catenin for translocation in hepatocytes is the Met-beta-catenin complex, which appears to be independent of the E-cadherin-beta-catenin complex. To test the functionality of this association, we used a dominant-negative Met expression system that expresses only the extracellular and transmembrane regions of the beta-subunit of Met. A loss of Met-beta-catenin association resulted in abrogation of nuclear translocation of beta-catenin upon HGF stimulation. This event is tyrosine phosphorylation dependent, and the association of Met and beta-catenin is crucial for this event. We conclude that the HGF causes similar redistribution of beta-catenin as Wnt-1 in the hepatocytes and that this effect is attributable to subcellular association of Met and beta-catenin. The intracellular kinase domain of Met is essential for tyrosine phosphorylation and nuclear translocation of beta-catenin. Part of the multifunctionality of HGF might be attributable to nuclear beta-catenin and the resulting target gene expression.

Nitric oxide: a signaling molecule against mitochondrial permeability transition- and pH-dependent cell death after reperfusion.

Reperfusion of ischemic tissue can precipitate cell death. Much of this cell killing is related to the return of physiological pH after the tissue acidosis of ischemia. The mitochondrial permeability transition (MPT) is a key mechanism contributing to this pH-dependent reperfusion injury in hepatocytes, myocytes, and other cell types. When ATP depletion occurs after the MPT, necrotic cell death ensues. If ATP levels are maintained, at least in part, the MPT initiates apoptosis caused by mitochondrial swelling and release of cytochrome c and other proapoptotic factors. Cyclosporin A and acidotic pH inhibit opening of permeability transition pores and protect cells against oxidative stress and ischemia/reperfusion injury, whereas Ca(2+), mitochondrial reactive oxygen species, and pH above 7 promote mitochondrial inner membrane permeabilization. Reperfusion with nitric oxide (NO) donors also blocks the MPT via a guanylyl cyclase and protein kinase G-dependent signaling pathway, which in turn prevents reperfusion-induced cell killing. In isolated mitochondria, a combination of cGMP, cytosolic extract, and ATP blocks the Ca(2+)-induced MPT, an effect that is reversed by protein kinase G inhibition. Thus, NO prevents pH-dependent cell killing after ischemia/reperfusion by a guanylyl cyclase/cGMP/protein kinase G signaling cascade that blocks the MPT.

Effect of oxygen concentration on viability and metabolism in a fluidized-bed bioartificial liver using ³¹P and ¹³C NMR spectroscopy.

Many oxygen mass-transfer modeling studies have been performed for various bioartificial liver (BAL) encapsulation types; yet, to our knowledge, there is no experimental study that directly and noninvasively measures viability and metabolism as a function of time and oxygen concentration. We report the effect of oxygen concentration on viability and metabolism in a fluidized-bed NMR-compatible BAL using in vivo ³¹P and ¹³C NMR spectroscopy, respectively, by monitoring nucleotide triphosphate (NTP) and ¹³C-labeled nutrient metabolites, respectively. Fluidized-bed bioreactors eliminate the potential channeling that occurs with packed-bed bioreactors and serve as an ideal experimental model for homogeneous oxygen distribution. Hepatocytes were electrostatically encapsulated in alginate (avg. diameter, 500 µm; 3.5×107 cells/mL) and perfused at 3 mL/min in a 9-cm (inner diameter) cylindrical glass NMR tube. Four oxygen treatments were tested and validated by an in-line oxygen electrode: (1) 95:5 oxygen:carbon dioxide (carbogen), (2) 75:20:5 nitrogen:oxygen:carbon dioxide, (3) 60:35:5 nitrogen:oxygen:carbon dioxide, and (4) 45:50:5 nitrogen:oxygen:carbon dioxide. With 20% oxygen, ß-NTP steadily decreased until it was no longer detected at 11 h. The 35%, 50%, and 95% oxygen treatments resulted in steady ß-NTP levels throughout the 28-h experimental period. For the 50% and 95% oxygen treatment, a ¹³C NMR time course (∼5 h) revealed 2-¹³C-glycine and 2-¹³C-glucose to be incorporated into [2-¹³C-glycyl]glutathione (GSH) and 2-¹³C-lactate, respectively, with 95% having a lower rate of lactate formation. ³¹P and ¹³C NMR spectroscopy is a noninvasive method for determining viability and metabolic rates. Modifying tissue-engineered devices to be NMR compatible is a relatively easy and inexpensive process depending on the bioreactor shape.

Inhibition of mitochondrial respiration as a source of adaphostin-induced reactive oxygen species and cytotoxicity.

Adaphostin is a dihydroquinone derivative that is undergoing extensive preclinical testing as a potential anticancer drug. Previous studies have suggested that the generation of reactive oxygen species (ROS) plays a critical role in the cytotoxicity of this agent. In this study, we investigated the source of these ROS. Consistent with the known chemical properties of dihydroquinones, adaphostin simultaneously underwent oxidation to the corresponding quinone and generated ROS under aqueous conditions. Interestingly, however, this quinone was not detected in intact cells. Instead, high performance liquid chromatography demonstrated that adaphostin was concentrated by up to 300-fold in cells relative to the extracellular medium and that the highest concentration of adaphostin (3000-fold over extracellular concentrations) was detected in mitochondria. Consistent with a mitochondrial site for adaphostin action, adaphostin-induced ROS production was diminished by >75% in MOLT-4 rho(0) cells, which lack mitochondrial electron transport, relative to parental MOLT-4 cells. In addition, inhibition of oxygen consumption was observed when intact cells were treated with adaphostin. Loading of isolated mitochondria to equivalent adaphostin concentrations caused inhibition of uncoupled oxygen consumption in mitochondria incubated with the complex I substrates pyruvate and malate or the complex II substrate succinate. Further analysis demonstrated that adaphostin had no effect on pyruvate or succinate dehydrogenase activity. Instead, adaphostin inhibited reduced decylubiquinone-induced cytochrome c reduction, identifying complex III as the site of inhibition by this agent. Moreover, adaphostin enhanced the production of ROS by succinate-charged mitochondria. Collectively, these observations demonstrate that mitochondrial respiration rather than direct redox cycling of the hydroquinone moiety is a source of adaphostin-induced ROS and identify complex III as a potential target for antineoplastic agents.

Isolated mouse liver mitochondria are devoid of glucokinase.

Glucokinase is a hexokinase isoform with low affinity for glucose that has previously been identified as a cytosolic enzyme. A recent report claims that glucokinase physically associates with liver mitochondria to form a multi-protein complex that may be physiologically important in apoptotic signaling [N.N. Danial, C.F. Gramm, L. Scorrano, C.Y. Zhang, S. Krauss, A.M. Ranger, S.R. Datta, M.E. Greenberg, L.J. Licklider, B.B. Lowell, S.P. Gygi, S.J. Korsmeyer, Nature 424 (2003) 952-956]. Here, we re-examined the association of glucokinase with isolated mouse liver mitochondria. When glucokinase activity was measured by coupled enzyme assay, robust activity was present in whole liver homogenates and their 9500 g supernatants (cytosol), but activity in the purified mitochondrial fraction was below detection (<0.2% of homogenate). Furthermore, addition of 45 mM glucose in the presence of ATP did not increase mitochondrial respiration, indicating the absence of ADP formation by glucokinase or any other hexokinase isoform. Immunoblots of liver homogenates and cytosol revealed strong glucokinase bands, but no immunoreactivity was detected in mitochondria. In conclusion, mouse liver mitochondria lack measurable glucokinase. Thus, functional linkage of glucokinase to mitochondrial metabolism and apoptotic signaling is unlikely to be mediated by the physical association of glucokinase with mitochondria.

Nitric oxide protects rat hepatocytes against reperfusion injury mediated by the mitochondrial permeability transition.

We investigated the effects of nitric oxide (NO) on hepatocellular killing after simulated ischemia/reperfusion and characterized signaling factors triggering cytoprotection by NO. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 hours and reoxygenated at pH 7.4 for 2 hours. During reoxygenation, some hepatocytes were exposed to combinations of NO donors (S-nitroso-N-acetylpenicillamine [SNAP] and others), a cGMP analogue (8-bromoguanosine-3,5-cGMP [8-Br-cGMP]), and a cGMP-dependent protein kinase inhibitor (KT5823). Cell viability was determined by way of propidium iodide fluorometry. Inner membrane permeabilization and mitochondrial depolarization were monitored by confocal microscopy. SNAP, but not oxidized SNAP, increased cGMP during reperfusion and decreased cell killing. Other NO donors and 8-Br-cGMP also prevented cell killing. Both guanylyl cyclase and cGMP-dependent kinase inhibition blocked the cytoprotection of NO. However, 5-hydroxydecanoate and diazoxide- mitochondrial K(ATP) channel modulators-did not affect NO-dependent cytoprotection or reperfusion injury. During reoxygenation, confocal microscopy showed mitochondrial repolarization, followed by depolarization, inner membrane permeabilization, and cell death. In the presence of either SNAP or 8-Br-cGMP, mitochondrial repolarization was sustained after reperfusion preventing inner membrane permeabilization and cell death. In isolated rat liver mitochondria, a cGMP analogue in the presence of a cytosolic extract and adenosine triphosphate blocked the Ca(2+)-induced mitochondrial permeability transition (MPT), an effect that was reversed by KT5823. In conclusion, NO prevents MPT-dependent necrotic killing of ischemic hepatocytes after reperfusion through a guanylyl cyclase and cGMP-dependent kinase signaling pathway, events that may represent the target of NO cytoprotection in preconditioning.

Differential mitogenic effects of single chain hepatocyte growth factor (HGF)/scatter factor and HGF/NK1 following cleavage by factor Xa.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF.

A mechanism of cell survival: sequestration of Fas by the HGF receptor Met.

Death receptors such as Fas are present in a variety of organs including liver and play an important role in homeostasis. What prevents these harmful receptors from forming homooligomers, clustering, and initiating the apoptotic pathway is not known. Here, we report the discovery of a cell survival mechanism by which Met, a growth factor receptor tyrosine kinase, directly binds to and sequesters the death receptor Fas in hepatocytes. This interaction prevents Fas self-aggregation and Fas ligand binding, thus inhibiting Fas activation and apoptosis. Our results describe a direct link between growth factor tyrosine kinase receptors and death receptors to establish a novel paradigm in growth regulation.

Beta-catenin antisense studies in embryonic liver cultures: role in proliferation, apoptosis, and lineage specification.

BACKGROUND & AIMS: Wnt/beta-catenin pathway activation occurs during liver growth in hepatoblastomas, hepatocellular cancers, and liver regeneration. The aim of this study was to investigate the role of beta-catenin, a key component of the Wnt pathway, in liver development as well as its normal distribution in developing liver. METHODS: Embryonic liver cultures and beta-catenin antisense phosphorodiamidate morpholino oligomer (PMO) were used to elucidate the role of beta-catenin in liver development. Livers from embryos at 10 days of gestational development were cultured in the presence of antisense or control PMO for 72 hours and analyzed. RESULTS: Beta-catenin shows stage-specific localization and distinct distribution compared with known markers in developing liver. A substantial decrease in beta-catenin protein was evident in the organs cultured in the presence of antisense. Beta-catenin inhibition decreased cell proliferation and increased apoptosis in these organ cultures. Presence of antisense resulted in loss of CK19 immunoreactivity of the bipotential stem cells. Beta-catenin inhibition also promoted c-kit immunoreactivity of the hepatocytes. CONCLUSIONS: We conclude that the PMO antisense to beta-catenin effectively inhibits synthesis of its protein. Beta-catenin modulates cell proliferation and apoptosis in developing liver. It may play a significant role in early biliary lineage commitment of the bipotential stem cells and also seems to be important in hepatocyte maturation.