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© 2019 by the authors., Fatty acids and glucose are the main bioenergetic substrates in mammals. Impairment of mitochondrial fatty acid oxidation causes mitochondrial myopathy leading to decreased physical performance. Here, we report that haploinsufficiency of ADCK2, a member of the aarF domain-containing mitochondrial protein kinase family, in human is associated with liver dysfunction and severe mitochondrial myopathy with lipid droplets in skeletal muscle. In order to better understand the etiology of this rare disorder, we generated a heterozygous Adck2 knockout mouse model to perform in vivo and cellular studies using integrated analysis of physiological and omics data (transcriptomics–metabolomics). The data showed that Adck2+/− mice exhibited impaired fatty acid oxidation, liver dysfunction, and mitochondrial myopathy in skeletal muscle resulting in lower physical performance. Significant decrease in Coenzyme Q (CoQ) biosynthesis was observed and supplementation with CoQ partially rescued the phenotype both in the human subject and mouse model. These results indicate that ADCK2 is involved in organismal fatty acid metabolism and in CoQ biosynthesis in skeletal muscle. We propose that patients with isolated myopathies and myopathies involving lipid accumulation be tested for possible ADCK2 defect as they are likely to be responsive to CoQ supplementation., This research was supported by grants from the Spanish Ministry of Health, Instituto de Salud Carlos III (ISCIII), PI17/01286 and Andalussian Government Excellence grant P12-CTS-943 to P.N., FIS PI17/00190 to R.A., by grants from Istituto di Ricerca Pediatrica Città della Speranza and Telethon Italy (GGP13222 and GGP14187c) to L.S., and the Intramural Research Program of National Institute on Aging, National Institutes of Health (NIA/NIH) (M.A.A., M.B. and R.d.C.). L.V.-F. doctoral thesis was directed by C.S.-O. I.N.-E. and A.d.F. were postdoctoral fellows at National Institute on Aging, National Institutes of Health (NIA/NIH).

Cytochrome b5 reductase 3 (CYB5R3) is a membrane-bound NADH-dependent redox enzyme anchored to the mitochondrial outer membrane, endoplasmic reticulum, and plasma membrane. Recessive hereditary methaemoglobinaemia (RHM) type II is caused by CYB5R3 deficiency and is an incurable disease characterized by severe encephalopathy with mental retardation, microcephaly, generalized dystonia, and movement disorders. Currently, the etiology of type II RHM is poorly understood and there is no treatment for encephalopathy associated with this disease. Defective CYB5R3 leads to defects in the elongation and desaturation of fatty acids and cholesterol biosynthesis, which are conventionally linked with neurological disorders of type II RHM. Nevertheless, this abnormal lipid metabolism cannot explain all manifestations observed in patients. Current molecular and cellular studies indicate that CYB5R3 deficiency has pleiotropic tissue effects. Its localization in lipid rafts of neurons indicates its role in interneuronal contacts and its presence in caveolae of the vascular endothelial membrane suggests a role in the modulation of nitric oxide diffusion. Its role in aerobic metabolism and oxidative stress in fibroblasts, neurons, and cardiomyocytes has been reported to be due to its ability to modulate the intracellular ratio of NAD+/NADH. Based on the new molecular and cellular functions discovered for CYB5R3 linked to the plasma membrane and mitochondria, the conventional conception that the cause of type II RHM is a lipid metabolism disorder should be revised. We hypothesized that neurological symptoms of the disease could be caused by disorders in the synapse, aerobic metabolism, and/or vascular homeostasis rather than in disturbances of lipid metabolism., This work has been funded by the Instituto de Salud Carlos III FIS PI17-01286 grant. Authors were also funded by the Andalusian Government BIO177 research group.

et al., Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3′-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ biosynthesis., This research was supported by grants from the Spanish Ministry of Health, Instituto de Salud Carlos III (ISCIII), FIS PI14–01962 to PN, the Spanish Ministry of Economy and Competitively, SAF2013–41945-R to JMC, and the Intramural Research Program of the NIA, NIH (KA, MG).

We previously demonstrated that extracellular calcium regulates vitamin D receptor (VDR) expression by parathyroid cells. Since the calcimimetic R-568 potentiates the effects of calcium on the calcium-sensing receptor, it was hypothesized that administration of R-568 may result in increased VDR expression in parathyroid tissue. In vitro studies of the effect of R-568 on VDR mRNA and protein were conducted in cultures of whole rat parathyroid glands and human hyperplastic parathyroid glands. In vivo studies in Wistar rats examined the effect of R-568 and calcitriol alone and in combination. Incubation of rat parathyroid glands in vitro with R-568 (0.001–1 μM) resulted in a dose-dependent decrease in parathyroid hormone (PTH) secretion and an increase in VDR expression (mean ± SE). Incubation in 1 mM calcium + 0.001 μM R-568 elicited an increase in VDR mRNA (306 ± 46%) similar to the maximum increase detected with 1.5 mM calcium (330 ± 42%). In vivo, VDR mRNA was increased after administration of R-568 (168 ± 9%, P < 0.001 vs. control) or calcitriol (198 ± 16%, P < 0.001 vs. control). Treatment with R-568 also increased VDR protein in normal rat parathyroid glands and in human parathyroid glands with diffuse, but not nodular, hyperplasia. In conclusion, the present study shows that the calcimimetic R-568 exerts a stimulatory effect on VDR expression in the parathyroid glands of study models and provides additional evidence for the use of calcimimetics in the treatment of secondary hyperparathyroidism.

In the last few years, it has become clear that the processes of tumor angiogenesis, metastasis and invasiveness are highly dependent on components of the blood coagulation cascade. One of the key proteins in coagulation is tissue factor (TF). In addition, TF is also known as a mediator of intracellular signaling events that can alter gene expression patterns and cell behavior. TF significantly participates in tumor-associated angiogenesis and its expression levels have been correlated with the metastatic potential of many types of hematological malignancies. Signaling pathways initiated by both, tissue factor-activated factor VII (TF-FVII(a)) protease activation of protein-activated receptors (PARs), and phosphorylation of the TF-cytoplasmic domain, appear to regulate these tumoral functions. Advances in antiangiogenic therapies and preclinical studies with TF-targeted therapeutics are hopeful in the control of tumor growth and metastasis, but continued studies on the regulation of TF are still needed. In the last few years, the use of approaches of functional genomics and proteomics has allowed the discovery of new proteins involved in the origin of the neoplasia and their participation in the development of the disease. This review attempts to establish a cellular and molecular causal link between cancer coagulopathy, angiogenesis and tumor progression in hematological malignancies.

The main goal of this study was to analyze, using proteomic techniques, changes in protein expression of acute myeloid leukemia (AML) cells that could give insights into a better early prognosis for tumor pathophysiology. Proteomic analysis of different subtypes of AML cells was carried out using 2-DE and MALDI-TOF PMF analysis. Proteins identified as more significantly altered between the different AMLs belonged to the group of suppressor genes, metabolic enzymes, antioxidants, structural proteins and signal transduction mediators. Among them, seven identified proteins were found significantly altered in almost all the AML blast cells analyzed in relation to normal mononuclear blood cells: alpha-enolase, RhoGDI2, annexin A10, catalase, peroxiredoxin 2, tromomyosin 3, and lipocortin 1 (annexin 1). These differentially expressed proteins are known to play important roles in cellular functions such as glycolysis, tumor suppression, apoptosis, angiogenesis and metastasis, and they might contribute to the adverse evolution of the disease. Proteomic analysis has identified for the first time novel proteins that may either help to form a differential prognosis or be used as markers for disease outcome, thus providing potential new targets for rational pathogenesis-based therapies of AML.

Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. Nitric oxide (NO) mediates PGE1 protection against D-galactosamine (D-GalN)-induced cell death. Nuclear factor kappa-B (NF-kappaB) plays a protective role in different experimental models of cell death. We investigated if NF-kappaB was responsible for inducible nitric oxide synthase (iNOS) expression and cytoprotection induced by PGE1 against D-GalN cell death in cultured hepatocytes. Rat hepatocytes were isolated following the classical method of collagenase perfusion of liver. A kinetic study of cell death, NF-kappaB activation, mRNA and protein iNOS expression, and NO production was carried in hepatocytes treated with D-GalN (5 mM) in the presence or absence of PGE1 (1 microM) administered 2 h before the hepatotoxin. A proteasome inhibitor was used to evaluate the role of NF-kappaB activation in our experimental conditions. PGE1 protection against D-GalN-induced cell death was associated with its capacity to rapidly enhance NF-kappaB activation, mRNA and protein iNOS expression, and NO production in D-GalN-treated hepatocytes. The inhibition of NF-kappaB activation abolished iNOS expression and cell protection by PGE1 in hepatocytes treated with the hepatotoxin. The present study shows that the cytoprotection by PGE1 against D-GalN-induced apoptosis was related to NF-kappaB-dependent iNOS expression.

Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical manifestations of liver dysfunction. Nitric oxide (NO) has been shown to exert a protective or noxious effect in different experimental models of liver injury. The aim of the present study was to investigate the role of NO during PGE1 protection against d -galactosamine ( d -GalN) citotoxicity in cultured hepatocytes. PGE1 was preadministered to d -GalN-treated hepatocytes. The role of NO in our system was assessed by iNOS inhibition and a NO donor. Different parameters related to apoptosis and necrosis, NO production such as nitrite + nitrate (NOx) release, iNOS expression, and NF-κB activation in hepatocytes were evaluated. The inhibition of iNOS reduced apoptosis induced by d -GalN in hepatocytes. PGE1 protection against d -GalN injury was associated with its capacity to reduce iNOS expression and NO production induced by d -GalN. Nevertheless, iNOS inhibition showed that protection by PGE1 was also mediated by NO. Low concentrations of a NO donor reduced d -GalN injury with a decrease in the extracellular NOx concentration. High concentrations of the NO donor enhanced NOx concentration and increased cell death by d -GalN. The present study suggests that low NO production induced by PGE1 preadministration reduces d -GalN-induced cell death through its capacity to reduce iNOS expression and NO production caused by the hepatotoxin.

BACKGROUND: Prostaglandin E1(PGE1) reduces cell death in experimental and clinical liver dysfunction.OBJECTIVES: Whether PGE1protects against d-galactosamine (D-GalN)-associated hepatocyte cell death by the regulation of tumour necrosis factor-alpha (TNF-alpha) and/or nitric oxide (NO) in hepatocytes or cocultured Kupffer cells was examined.METHODS: Anti-TNF-alpha antibodies were used to evaluate the role of TNF-alpha during d-GalN cytotoxicity and its protection by PGE1in cocultured hepatocytes and Kupffer cells. Cell apoptosis and necrosis were assessed by DNA fragmentation and lactate dehydrogenase release, respectively. Nitrite+nitrate (NOx), as NO end products, and TNF-alpha concentrations were measured in the culture medium. The role of NO was determined by measuring inducible NO synthase (iNOS) expression and the effect of its inhibition during d-GalN cytotoxicity and its protection by PGE1.RESULTS: d-GalN enhanced hepatocyte cell death associated with high TNF-alpha and NOx levels in a culture medium. Anti-TNF-alpha and iNOS inhibition suggested that TNF-alpha was mediating apoptosis, but not necrosis, through the stimulation of NO production. The antiapoptotic activity of PGE1was associated with a reduction of NO production, but was blocked by iNOS inhibition. This apparent contradiction was explained by the ability of PGE1to enhance iNOS expression shortly after its administration and inhibit it later during d-GalN treatment. Anti-TNF-alpha antibodies did not reduce the exacerbation ofD-GalN-associated cell death in hepatocytes by cocultured Kupffer cells.CONCLUSION: TNF-alpha mediatesD-GalN-induced apoptosis via NO production in cultured hepatocytes. The protective effect of PGE1againstD-GalN-induced apoptosis is probably through the induction of low iNOS expression that was followed by a reduction of iNOS expression and NO production induced by the hepatotoxin. The exacerbation of hepatocyte cell death by Kupffer cells was not related to TNF-alpha and NO.

D-galactosamine (D-GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE1 treatment reduced necrosis and apoptosis induced by D-GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by D-GalN (0-40mM). Also, the present study investigated if PGE1 (1 microM) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H2O2), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of D-GalN concentrations (2.5-10mM) induced apoptosis in association with a moderate oxidative stress. High D-GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with D-GalN (2.5-10 mM)-treated cells. Although PGE1 reduced apoptosis induced by D-GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.

Cytochrome b5 reductases (CYB5R) are required for the elongation and desaturation of fatty acids, cholesterol synthesis and mono-oxygenation of cytochrome P450 enzymes, all of which are associated with protection against metabolic disorders. However, the physiological role of CYB5R in the context of metabolism, healthspan and aging remains ill-defined. We generated CYB5R-overexpressing flies (CYB5R-OE) and created a transgenic mouse line overexpressing CYB5R3 (CYB5R3-Tg) in the C57BL/6J background to investigate the function of this class of enzymes as regulators of metabolism and age-associated pathologies. Gender- and/or stage-specific induction of CYB5R, and pharmacological activation of CYB5R with tetrahydroindenoindole extended fly lifespan. Increased expression of CYB5R3 was associated with significant improvements in several metabolic parameters that resulted in modest lifespan extension in mice. Diethylnitrosamine-induced liver carcinogenesis was reduced in CYB5R3-Tg mice. Accumulation of high levels of long-chain polyunsaturated fatty acids, improvement in mitochondrial function, decrease in oxidative damage and inhibition of chronic pro-inflammatory pathways occurred in the transgenic animals. These results indicate that CYB5R represents a new target in the study of genes that regulate lipid metabolism and healthspan.

6 páginas, 3 figuras.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License., Aging results from a complex and not completely understood chain of processes that are associated with various negative metabolic consequences and ultimately leads to senescence and death. The intracellular ratio of pyridine nucleotides (NAD+/NADH), has been proposed to be at the center stage of age-related biochemical changes in organisms, and may help to explain the observed influence of calorie restriction and energy-sensitive proteins on lifespan in model organisms. Indeed, the NAD+/NADH ratios affect the activity of a number of proteins, including sirtuins, which have gained prominence in the aging field as potential mediators of the beneficial effects of calorie restriction and mediating lifespan. Here we review the activities of a redox enzyme (NQR1 in yeast and CYB5R3 in mammals) that also influences the NAD+/NADH ratio and may play a regulatory role that connects aerobic metabolism with aging., This paper has been partially supported by Spanish FIS Grant PI080500, NIH Grant 1R01AG028125-01A1, and the Intramural Research Program of the NIH, National Institute on Aging.

BACKGROUND: Coenzyme Q (CoQ) is a lipophilic antioxidant that is synthesized by a mitochondrial complex integrated by at least ten nuclear encoded COQ gene products. CoQ increases cell survival under different stress conditions, including mitochondrial DNA (mtDNA) depletion and treatment with cancer drugs such as camptothecin (CPT). We have previously demonstrated that CPT induces CoQ biosynthesis in mammal cells. METHODOLOGY/PRINCIPAL FINDINGS: CPT activates NF-kappaB that binds specifically to two kappaB binding sites present in the 5'-flanking region of the COQ7 gene. This binding is functional and induces both the COQ7 expression and CoQ biosynthesis. The inhibition of NF-kappaB activation increases cell death and decreases both, CoQ levels and COQ7 expression induced by CPT. In addition, using a cell line expressing very low of NF-kappaB, we demonstrate that CPT was incapable of enhancing enhance both CoQ biosynthesis and COQ7 expression in these cells. CONCLUSIONS/SIGNIFICANCE: We demonstrate here, for the first time, that a transcriptional mechanism mediated by NF-kappaB regulates CoQ biosynthesis. This finding contributes new data for the understanding of the regulation of the CoQ biosynthesis pathway.

The hallmark of acute promyelocytic leukaemia (APL) is the reciprocal translocation t(15;17), which leads to the expression of the promyelocytic leukaemia/retinoic acid receptor α (PML/RARα) fusion protein and a cell differentiation blockade at the promyelocytic stage. PML/RARα is directly targeted by all-trans-retinoic acid (ATRA), which degrades the oncoprotein and induces complete remission of malignancies. The aberrant function of PML/RARα, together with the constitutive activation of the mitogen-activated protein/extracellular signal-regulated kinase (MEK/ERK) signalling pathway, regulates the ability of haematopoietic cells to proliferate, differentiate, and escape from apoptotic episodes. The role of the MEK/ERK pathway in PML/RARα expression, differentiation, proliferation and apoptosis in APL cells was analysed using specific MEK inhibitors. The blockade of MEK/ERK pathway resulted in caspase-dependent degradation of PML/RARα, and attenuation of the cell differentiation induction. To our knowledge, this is the first report to show that PML/RARα was suppressed by MEK/ERK inhibition, through a mechanism dependent on caspase activation. ATRA co-operated with MEK inhibitor to increase degradation of PML/RARα and exhibited a convergence point in caspase activation with MEK inhibitors. Taken together, our data suggest a new role of MEK/ERK pathway in the pathogenesis of APL, thus supporting the use of MEK/ERK inhibitors as an efficient therapeutic strategy for this haematological malignancy., This work was supported by grants from the ‘Junta de Andalucia’ (Exp. 0024/05 and exp. 0060/05), the ‘Fondo de Investigacion Sanitaria’ (FIS PI050910 and PI041291) and The Josep Carreras International Leukaemia Foundation (FIJC-06/ESP) of Spain.

The Flt3 receptor tyrosine kinase is a critical mediator in the pathogenesis of acute myeloid leukaemia (AML). Flt3-activating mutations have been associated with poor prognosis and decreased overall survival of AML patients, thus Flt3 constitutes an ideal target for drug treatment of such disease. Unfortunately, the monotherapy with small-molecule tyrosine kinase inhibitors in clinical trials shows that remission is not permanent, presumably by resistance of Flt3 mutants to inhibitors. An alternative approach for treatment is based on the cooperation between Flt3 and additional intracellular pathways for AML transformation in some patients. Thus, the inhibition of both Flt3 and such pathways may be exploited for successful treatment of the disease. We investigated the importance of Flt3-activating mutations for the constitutive activation of intracellular pathways in primary AML cells, and their effect on cell survival. We found that the main compounds involved in the differentiation, proliferation and survival of AML (MAPK/AKT/STAT) were constitutively activated. However, only four samples showed internal tandem duplications (ITDs) for Flt3. Surprisingly, contrary to previous reports, we found that inhibition of ITD/Flt3 activity did not prevent the phosphorylation of ERK, STAT5 or Akt in some primary AML cells. In parallel, we found that in these cells, Flt3 and ERK or Akt cooperate to regulate cell survival. Our results support the hypothesis that the optimal therapeutic treatment of AML may require not only the oncogenic tyrosine kinase, but also the appropriate combination of different specific inhibitors, thus providing a more effective approach to reverse leukaemogenesis. Thus, we propose that each AML patient should have an individually tailored combination treatment., This work was supported by grants from the Fondo de Investigacion Sanitaria (PI050910 and PI041291) and the Junta de Andalucía (0024/2005) of Spain.

Cyclin D3 deregulation has recently been reported in bladder cancer but its prognostic significance remains uncertain. A cohort of 159 patients with stage Ta or T1 primary bladder tumours was investigated to determine the significance of cyclin D3 expression in association with other G1-S phase regulators of the cell cycle (p53, p21Waf1, p27kip1, cyclin D1), including tumour proliferation (ki67-MIB1); its association with conventional clinicopathological parameters; and the relationship between cyclin D3 and loss of heterozygosity (LOH) at the 9p21 (p16INK4a locus) chromosome region. The end point of the study was progression-free survival. Cyclin D3, other G1-S phase regulators, and tumour proliferation were investigated by immunohistochemistry and measured by the grid-counting method. To validate the immunohistochemical expression, cyclin D3 was additionally assessed by western blotting in selected cases. LOH at the 9p21 chromosome region (marker D9S171) was assessed in 125 cases using an AB Prism 310 genetic analyser and a set of microsatellite fluorescence-labelled primers. Cyclin D3 overexpression was related to larger tumour size (5 cm; p0.0001) and high tumour proliferation (10%; p = 0.025). Mean cyclin D3 expression increased with 2004 WHO grading categories in stage Ta (p = 0.035, ANOVA) and stage T1 (p = 0.047, t test) tumours. Cyclin D3 was not related to other clinicopathological parameters, G1-S phase modulators, or 9p21 LOH. Cox's multivariate analysis selected cyclin D3 as an independent predictor of progression-free survival (p = 0.0012, relative risk (RR) = 5.2366) together with tumour size (p = 0.0115, RR = 4.4442) and cyclin D1 (p = 0.0065, RR = 3.3023). Cyclin D3 expression had the highest risk ratio. Our results suggest that expression of cyclin D3 is relevant to the progression-free survival of patients with Ta/T1 bladder carcinomas.

The antiphospholipid syndrome (APS) is characterized by thrombosis or pregnancy morbidity in the presence of antiphospholipid autoantibodies (aPL). aPL are a heterogeneous family of autoantibodies with diverse cross-reactivities whose origin and role have not been fully elucidated. Many of the autoantibodies associated with APS are directed against phospholipid-binding plasma proteins, such as β2-GPI and prothrombin, or phospholipid-protein complexes. The mechanisms by which aPL cause thrombosis are not completely understood. There is no unique mechanism able to explain all symptoms associated with the presence of aPL. Different theories have been proposed, including the effect of aPL on endothelial cells, monocytes, and platelets. aPL are able to recognize, injure, or activate cultured vascular endothelial cells. Cultured endothelial cells incubated with aPL express increased levels of cell adhesion molecules and tissue factor (TF), an effect mediated by β2-GPI, and may promote inflammation and thrombosis. Overexpression of TF has been also shown in monocytes in vitro and ex vivo. TF is the major initiator of coagulation in vivo; thus, its dysregulation may be one of the most important contributors to thrombosis. Effects of aPL upon platelets are not completely elucidated. aPL bind anionic phospholipid but they are normally in the inner side of cell membranes. When platelets are activated by different agonists, anionic phospholipids are exposed. There is some evidence showing that activated platelets are present in aPL-positive patients. Increased levels of β-thrombomodulin, and microvesicle formation seem to support this hypothesis. Activated platelets may contribute to thrombosis by persistent exposure of a procoagulant surface.

Objective Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL). In patients with primary APS, expression of tissue factor (TF) on the surface of monocytes is increased, which may contribute to thrombosis in these patients. However, the intracellular mechanisms involved in aPL-mediated up-regulation of TF on monocytic cells are not understood. This study was undertaken to investigate the intracellular signals induced by aPL that mediate TF activation in monocytes from APS patients. Methods We analyzed, both in vivo and in vitro, aPL interactions with proteins that have signaling functions, including mitogen-activated protein kinases (MAP kinases) and NF-κB/Rel proteins. Results In vivo studies demonstrated significantly higher levels of both TF messenger RNA and TF protein in monocytes from APS patients compared with controls. At the molecular level, increased proteolysis of IκBα and activation of NF-κB were observed. Constitutive activation of both p38 and ERK-1 MAP kinases was also found. Treatment of normal monocytes with aPL activated ERK-1 and p38 MAP kinases, as well as the IκB/NF-κB pathway, in a dose-dependent manner. NF-κB activation and IκBα degradation induced by aPL were inhibited by the NF-κB inhibitor SN50 and the p38 MAP kinase inhibitor SB203580, thus suggesting crosstalk between these pathways. However, the MEK-1/ERK inhibitor PD98059 did not affect aPL-induced NF-κB binding activity. TF expression induced by aPL was significantly inhibited by combined treatment with the 3 inhibitors. Conclusion Our results suggest that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK-1/ERK proteins, and the p38 MAP kinase–dependent nuclear translocation and activation of NF-κB/Rel proteins.

Background and Aim: PGE1 reduces in vivo and in vitro D-galactosamine (D-GalN)-induced cell death in hepatocytes. The present study was undertaken to elucidate the intracellular pathway by which D-GalN induces cell death in cultured hepatocytes. In addition, we evaluated if PGE1 was able to modulate different parameters related to D-GalN-induced apoptosis in cultured rat hepatocytes. Methods: Hepatocytes were isolated from male Wistar rats (225–275 g) by the classical collagenase procedure. PGE1 (1 µM) was administered 2 h before D-GalN (5 mM) in primary culture of rat hepatocytes. Apoptosis was determined by DNA fragmentation and caspase-3, -6, -8 and -9 activation in hepatocytes. Caspase activation was evaluated by the detection of the related cleaved product and its associated activity. Cell necrosis was determined by the measurement of lactate dehydrogenase (LDH) activity in culture medium. To elucidate the role of mitochondria, we measured neutral (nSMase) and acid (aSMase) sphingomyelinase, as well as the expression of cytochrome c in mitochondria and cytoplasm fractions from D-GalN treated hepatocytes. Results: D-GalN induced caspase-3 activation and DNA fragmentation in hepatocytes. This apoptotic response was not associated with the activation of caspase-6, -8 or -9. The use of specific inhibitors confirmed that only caspase-3 was involved in D-GalN-induced apoptosis. D-GalN did not modify nSMase and aSMase activities, nor mitochondrial cytochrome c release in hepatocytes. Conclusions: D-GalN induced apoptosis through caspase-3 activation but without modification of the activity of caspase-6, -8, -9, SMases or cytochrome c release. PGE1 appears to prevent D-GalN-induced apoptosis by a mitochondria-independent mechanism.

Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. We have previously shown that PGE1 preadministration protects against NO-dependent cell death induced by D-galactosamine (D-GalN) through a rapid increase of nuclear factor κB (NF-κB) activity, inducible NO synthase (NOS-2) expression, and NO production. The present study investigates whether PGE1-induced NO was able to abolish NF-κB activation, NOS-2 expression, and apoptosis elicited by D-GalN. Rat hepatocytes were isolated following the classical method of collagenase perfusion of liver. PGE1 (1 μmol/L) was administered 2 hours before D-GalN (5 mmol/L) in primary culture rat hepatocytes. PGE1 reduced inhibitor κBα degradation, NF-κB activation, NOS-2 expression, and apoptosis induced by D-GalN. The administration of an inhibitor of NOS-2 abolished the inhibitory effect of PGE1 on NF-κB activation and NOS-2 expression in D-GalN–treated hepatocytes. Transfection studies using different plasmids corresponding to the NOS-2 promoter region showed that D-GalN and PGE1 regulate NOS-2 expression through NF-κB during the initial stage of hepatocyte treatment. PGE1 was able to reduce the promoter activity induced by D-GalN. In addition, a NO donor reduced NOS-2 promoter activity in transfected hepatocytes. In conclusion, administration of PGE1 to hepatocytes produces low levels of NO, which inhibits its own formation during D-GalN–induced cell death through the attenuation of NF-κB–dependent NOS-2 expression. Therefore, a dual role for NO in PGE1-treated D-GalN–induced toxicity in hepatocytes is characterized by a rapid NO release that attenuates the late and proapoptotic NOS-2 expression. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2004;40:1295–1303.)

Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. Nitric oxide (NO) mediates PGE1 protection against D-galactosamine (D-GalN)-induced cell death. Nuclear factor kappa-B (NF-kappaB) plays a protective role in different experimental models of cell death. We investigated if NF-kappaB was responsible for inducible nitric oxide synthase (iNOS) expression and cytoprotection induced by PGE1 against D-GalN cell death in cultured hepatocytes. Rat hepatocytes were isolated following the classical method of collagenase perfusion of liver. A kinetic study of cell death, NF-kappaB activation, mRNA and protein iNOS expression, and NO production was carried in hepatocytes treated with D-GalN (5 mM) in the presence or absence of PGE1 (1 microM) administered 2 h before the hepatotoxin. A proteasome inhibitor was used to evaluate the role of NF-kappaB activation in our experimental conditions. PGE1 protection against D-GalN-induced cell death was associated with its capacity to rapidly enhance NF-kappaB activation, mRNA and protein iNOS expression, and NO production in D-GalN-treated hepatocytes. The inhibition of NF-kappaB activation abolished iNOS expression and cell protection by PGE1 in hepatocytes treated with the hepatotoxin. The present study shows that the cytoprotection by PGE1 against D-GalN-induced apoptosis was related to NF-kappaB-dependent iNOS expression.

Low extracellular calcium (Ca) stimulates parathyroid hormone (PTH) secretion and also increases the renal synthesis of calcitriol (CTR), which is known to decrease PTH production. This study began with the hypothesis that the parathyroid cell response to CTR may be modulated by extracellular Ca concentration through an effect on parathyroid cell vitamin D receptor (VDR). In the present study, rat parathyroid glands were incubated in low (0.6 mM) and high (1.5 mM) Ca concentration. The parathyroid VDRmRNA was higher in 1.5 than 0.6 mM Ca. Furthermore, this effect was not observed in incubated slices of kidney cortex and medulla, tissues which also possess both Ca and vitamin D receptors. Experiments were also performed to evaluate the effect of Ca on VDR expression in vivo. Male Wistar rats received intraperitoneal injections of CaCl(2) or a single intramuscular injection of EDTA to obtain 6 h of hypercalcemic (ionized Ca, 1.4 to 1.6 mM) or hypocalcemic (ionized Ca, 0.85 to 0.95 mM) clamp; a third group of rats was used as control. A small dose of CTR was administered to hypercalcemic rats to match the serum CTR levels of hypocalcemic rats. Parathyroid gland VDRmRNA and VDR protein were increased in hypercalcemic rats as compared with hypocalcemic rats. Increasing doses of CTR upregulated VDRmRNA and VDR only in hypercalcemic rats. Additional experiments showed that the decrease in VDR in hypocalcemic rats prevented the inhibitory effect of CTR on PTHmRNA. In conclusion, our study shows that extracellular Ca regulates VDR expression by parathyroid cells independently of CTR and that by this mechanism hypocalcemia may prevent the feedback of CTR on the parathyroids.

Hypocotyls of kidney beans (Phaseolus vulgaris L.) accumulated ascorbate after preincubation with a number of possible precursors, mainlyl-galactono-γ-lactone (l-GL) andl-gulono-γ-lactone. The increase in the intracellular ascorbate concentration was parallel to the high stimulation of thel-GL dehydrogenase (l-GLD) activity measured in vitro using l-GL as a substrate and cytochromec as an electron acceptor. Cell fractionation using a continuous linear Percoll gradient demonstrated that l-GLD is associated with mitochondria; therefore, pure mitochondria were isolated and subjected to detergent treatment to separate soluble from membrane-linked proteins. l-GLD activity was mainly associated with the detergent phase, suggesting that a membrane-intrinsic protein is responsible for the ascorbic acid biosynthetic activity. Subfractionation of mitochondria demonstrated that l-GLD is located at the inner membrane.