Matrix metalloproteinase 2 in reduced-size liver transplantation: beyond the matrix.

We studied the contribution of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) to the beneficial effects of preconditioning (PC) in reduced-size orthotopic liver transplantation (ROLT). We also examined the role of c-Jun N-terminal kinase (JNK) and whether it regulates MMP2 in these conditions. Animals were subjected to ROLT with or without PC and pharmacological modulation, and liver tissue samples were then analyzed. We found that MMP2, but notMMP9, is involved in the beneficial effects of PC in ROLT. MMP2 reduced hepatic injury and enhanced liver regeneration. Moreover, inhibition of MMP2 in PC reduced animal survival after transplantation. JNK inhibition in the PC group decreased hepatic injury and enhanced liver regeneration. Furthermore, JNK upregulated MMP2 in PC. In addition, we showed that Tissue inhibitors of matrix metalloproteinases 2 (TIMP2) was also upregulated in PC and that JNK modulation also altered its levels in ROLT and PC. Our results open up new possibilities for therapeutic treatments to reduce I/R injury and increase liver regeneration after ROLT, which are the main limitations in living-donor transplantation.

Switches in 6-phosphofructo-2-kinase isoenzyme expression during rat sperm maturation.

Here we analyzed Pfkfb3 and Pfkfb4 gene expression in rat testis development, isolated testicular cells and spermatozoa. Real time RT-PCR analysis during testis development showed the maximum expression of Pfkfb3 in pre-puber samples and of Pfkfb4 in adult samples. Western blot analysis showed that uPFK-2 protein, a product of Pfkfb3 gene, was present in all the cell types forming the seminiferous epithelium (Sertoli, interstitial and spermatogenic cells). In contrast, tPFK-2, a product of Pfkfb4 gene, was restricted to spermatogenic cells. Confocal analyses by indirect immunofluorescence also corroborated this expression pattern. Immunoblotting studies of isolated spermatozoa demonstrated the presence of uPFK-2 only in immature sperm and once spermatozoa became fully functional this isozyme was replaced by the testicular isozyme tPFK-2. Moreover, immunostaining confirmed that tPFK-2 was localized mainly in the acrosomal region of the sperm head and in the mid-piece of the flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found.

PFKFB3 gene silencing decreases glycolysis, induces cell-cycle delay and inhibits anchorage-independent growth in HeLa cells.

The high rate of glycolysis despite the presence of oxygen in tumor cells (Warburg effect) suggests an important role for this process in cell division. The glycolytic rate is dependent on the cellular concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), which, in turn, is controlled by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). The ubiquitous PFK-2 isoenzyme (uPFK-2, alternatively named UBI2K5 or ACG) coded by the pfkfb3 gene is induced by different stimuli (serum, progesterone, insulin, hypoxia, etc.) and has the highest kinase/phosphatase activity ratio amongst all PFK-2 isoenzymes discovered to date, which is consistent with its role as a powerful activator of glycolysis. uPFK-2 is expressed in brain, placenta, transformed cells and proliferating cells. In the present work, we analyze the impact of small interfering RNA (siRNA)-induced silencing of uPFK-2 on the inhibition of cell proliferation. HeLa cells treated with uPFK-2 siRNA showed a decrease in uPFK-2 RNA levels measured at 24h. uPFK-2 protein levels were severely depleted at 48-72h when compared with cells treated with an unrelated siRNA, correlating with decreased glycolytic activity, Fru-2,6-P2, lactate and ATP concentrations. These metabolic changes led to reduced viability, cell-cycle delay and an increase in the population of apoptotic cells. Moreover, uPFK-2 suppression inhibited anchorage-independent growth. The results obtained highlight the importance of uPFK-2 on the regulation of glycolysis, on cell viability and proliferation and also on anchorage-independent growth. These data underscore the potential for uPFK-2 as an effective tumor therapeutic target.

Repression of SOX6 transcriptional activity by SUMO modification.

SOX6 plays key functions in several developmental processes, including neurogenesis and skeleton formation. In this report, we show that SOX6 is modified in vitro and in vivo by small ubiquitin-related modifier (SUMO) on two distinct sites. Mutation of both sites abolished SOX6 sumoylation and increased SOX6 transcriptional activity. SUMO dependent repression of SOX6 transcription was promoted by UBC9 whereas siRNA to UBC9, cotransfection of inactive UBC9 or a SUMO protease increased SOX6 transcriptional activity. Furthermore, co-expression of SOX6 with SUMO2 results in the appearance of SOX6 in a punctate nuclear pattern that colocalized with promyelocytic leukemia protein, which was partially abolished by mutations in SOX6 sumoylation sites.

How ischaemic preconditioning protects small liver grafts.

Interleukin-1 (IL-1) and transforming growth factor-beta (TGFbeta) are key inhibitors of hepatocyte proliferation after hepatectomy. IL-1 inhibition by heat shock proteins (HSPs) has been reported in inflammatory processes. A recent study indicated the benefits of ischaemic preconditioning in reduced-size orthotopic liver transplantation (ROLT). The present study examined: (a) the effect of ischaemic preconditioning on IL-1 and TGFbeta in ROLT; (b) whether preconditioning protects small liver grafts through HSP induction; and (c) whether the potential benefits of preconditioning on HSP is related to IL-1 inhibition. Our results, obtained with an IL-1 receptor antagonist, indicated the injurious effects of IL-1 in ischaemia-reperfusion (I/R) injury and established a relationship between IL-1 and growth factors. Thus, IL-1 reduced hepatocyte growth factor (HGF) and promoted TGFbeta release, thus contributing to the impaired liver regeneration associated with ROLT. Preconditioning inhibited IL-1 through nitric oxide (NO), thereby protecting against the injurious effects of IL-1. In addition, by another pathway independent of NO, preconditioning induced HSP70 and haem-oxygenase-1 (HO-1). HO-1 protected against I/R injury and liver regeneration, whereas the benefits resulting from HSP70 were mainly related to hepatocyte proliferation. These results suggest a mechanism that explains the effectiveness of preconditioning in ROLT. They suggest, too, that other strategies, in addition to preconditioning, that modulate IL-1 and/or HSPs could be considered in clinical situations requiring liver regeneration such as small liver grafts.

Copolymers of poly-L-lysine with serine and tryptophan form stable DNA vectors: implications for receptor-mediated gene transfer.

Inefficient gene transfer and poor stability in physiological medium are important shortcomings for receptor-mediated gene transfer vectors. Here, we evaluate vectors formulated with random copolymers of L-lysine/L-serine (3:1) and L-lysine/L-tryptophan (4:1), focusing on both their biophysical and functional characterization. By means of dynamic light scattering (DLS) and transmission electron microscopy (TEM), we demonstrate that poly-L-lysine (pK), poly-L-lysine-L-tryptophan (pKW) and poly-L-lysine-L-serine (pKS) are able to form compacted, small particles when mixed with plasmid DNA in the absence of salt. Upon dilution in physiological medium, copolymers of both lys/ser and lys/trp do not aggregate, in contrast with poly-L-lysine DNA complexes as determined by scattering, DLS and TEM measurements. Tight packing, as demonstrated by resistance to heparin, SDS and trypsin treatments, is also featured in tryptophan-containing complexes. Successful receptor-mediated endocytosis gene transfer using galactosylated copolymers into cells expressing the asiagloglycoprotein receptor correlated with lack of aggregation. Particles obtained using galactosylated poly-L-lysine-L-tryptophan (Gal-pKW) copolymer demonstrated specific receptor-mediated gene transfer since reporter gene activity dropped in the presence of an excess ligand in the culture medium during transfection. Although copolymers of galactosylated poly-L-lysine-L-serine (Gal-pKS) do not aggregate in the presence of salt, they are not able to internalize in a specific receptor-mediated endocytosis fashion. The introduction of bulky aromatic/hydrophobic (tryptophan) or hydrophillic (serine) moieties into the positively charged vectors allows the compacted particles to disperse into salt-containing medium avoiding salt-induced aggregation. Moreover, tryptophan-containing particles are able to mediate specific gene transfer via receptor-mediated endocytosis.

Receptor-mediated gene transfer vectors: progress towards genetic pharmaceuticals.

Although specific delivery to tissues and unique cell types in vivo has been demonstrated for many non-viral vectors, current methods are still inadequate for human applications, mainly because of limitations on their efficiencies. All the steps required for an efficient receptor-mediated gene transfer process may in principle be exploited to enhance targeted gene delivery. These steps are: DNA/vector binding, internalization, subcellular trafficking, vesicular escape, nuclear import, and unpacking either for transcription or other functions (i.e., antisense, RNA interference, etc.). The large variety of vector designs that are currently available, usually aimed at improving the efficiency of these steps, has complicated the evaluation of data obtained from specific derivatives of such vectors. The importance of the structure of the final vector and the consequences of design decisions at specific steps on the overall efficiency of the vector will be discussed in detail. We emphasize in this review that stability in serum and thus, proper bioavailability of vectors to their specific receptors may be the single greatest limiting factor on the overall gene transfer efficiency in vivo. We discuss current approaches to overcome the intrinsic instability of synthetic vectors in the blood. In this regard, a summary of the structural features of the vectors obtained from current protocols will be presented and their functional characteristics evaluated. Dissecting information on molecular conjugates obtained by such methodologies, when carefully evaluated, should provide important guidelines for the creation of effective, targeted and safe DNA therapeutics.

Induction of the Sry-related factor SOX6 contributes to bone morphogenetic protein-2-induced chondroblastic differentiation of C3H10T1/2 cells.

Chondrogenesis leads to the formation of mature cartilage and generates initial skeletal elements that serve as templates for endochondral bone formation. Bone morphogenetic proteins (BMPs) are involved in several developmental and organogenetic processes and have been identified as key regulators in chondrogenesis. In the present study we sought to determine the transcriptional mechanisms contributing to the induction of chondrogenic markers by BMP-2. Time-course studies with BMP-2-stimulated C3H10T1/2 cells showed a dose-dependent appearance of Alcian-blue-positive material and up-regulated expression of type-II collagen mRNA. This last effect required new protein synthesis because addition of cycloheximide completely blocked the induction of type-II collagen mRNA. A region encompassing the chondrocyte-specific enhancer, localized in intron I of type-II collagen alpha1 chain (Col2a1) gene, is sufficient to confer BMP-2-dependent transcriptional induction of type-II collagen gene expression. Analysis of the expression levels of chondrogenic Sry-type high-mobility group (HMG) box proteins (SOX) transcription factors demonstrated a time-dependent induction of Sox6 expression by BMP-2 that correlated with the appearance of BMP-2- induced protein complexes bound to the chondrocyte-specific enhancer. Preincubation of nuclear extracts with SOX6 and SOX9 antibodies markedly reduced the intensity of these bands. Forced expression of SOX6 mimicked the BMP-2 effect, whereas coexpression of SOX9 promoted a synergistic interaction between both factors in transcription from the chondrocyte-specific enhancer. Moreover, overexpression of a SOX6 mutated form, devoid of its high-mobility group domain, was sufficient to prevent transcriptional induction of the chondrocyte-specific enhancer by BMP-2. Taken together, these results indicate that SOX6 is an important downstream mediator of BMP-2 signaling in chondrogenesis.

Single-stranded DNA condensed with poly-L-lysine results in nanometric particles that are significantly smaller, more stable in physiological ionic strength fluids and afford higher efficiency of gene delivery than their double-stranded counterparts.

Nonviral gene transfer vectors have been actively studied in the past years in order to obtain structural entities with minimum size and defined shape. The final size of a gene transfer vector, which is compacted into unimolecular complexes, is directly proportional to the mass of the nucleic acid to be compacted. Thus, the purpose of this study was to assess the possibility of producing ssDNA vectors and their biophysical and biological characterization. We have obtained ssDNA/poly-L-lysine complexes that are significantly smaller than their double-stranded counterparts. We have also identified a lesser aggregative behavior of compacted single-stranded vs. double-stranded DNA vectors in the presence of physiological NaCl concentrations. Expression of compacted ssDNA is observed in hepatoma cell lines. Moreover, we have successfully delivered galactosylated ssDNA complexes into cells that express the asialoglycoprotein receptor via receptor-mediated endocytosis. The reduced size and biophysical behavior of ssDNA vectors may provide an advantage for transfection of eukaryotic cells.

Adenosine monophosphate-activated protein kinase mediates the protective effects of ischemic preconditioning on hepatic ischemia-reperfusion injury in the rat.

Hepatic ischemia-reperfusion (I/R) injury associated with liver transplantation and hepatic resections are an unresolved problem in the clinical practice. Preconditioning is known to preserve energy metabolism in liver during sustained ischemia, but the molecular mechanisms underlying this effect are still unclear. Different metabolic signals, including adenosine monophosphate (AMP) and nitric oxide (NO), have been implicated in preconditioning. AMP-activated protein kinase (AMPK) protects cells by acting as a low-fuel warning system, becoming switched on by adenosine triphosphate (ATP) depletion. NO synthesis is induced by AMPK in the heart during ischemia. The aim of this study was to investigate: 1) whether preconditioning induces AMPK activation; and 2) if AMPK activation leads to ATP preservation and reduced lactate accumulation during prolonged ischemia and its relationship with NO. Preconditioning activated AMPK and concomitantly reduced ATP degradation, lactate accumulation, and hepatic injury. The administration of an AMPK activator, AICAR, before ischemia simulated the benefits of preconditioning on energy metabolism and hepatic injury. The inhibition of AMPK abolished the protective effects of preconditioning. The effect of AMPK on energy metabolism was independent of NO because the inhibition of NO synthesis in the preconditioned group and the administration of the NO donor before ischemia, or to the preconditioned group with previous inhibition of AMPK, had no effect on energy metabolism. Both preconditioning and AICAR pretreatment, through AMPK activation, may be useful surgical and pharmacologic strategies aimed at reducing hepatic I/R injury.

The human ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFB3): promoter characterization and genomic structure.

A DNA fragment containing 1.5 kb of the 5'-flanking region of the human ubiquitous PFKFB3 gene, coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was cloned and its promoter activity was examined. The 5' flanking region contains a TATA box-like and GC-rich sequences, yielding several potential Specific protein (Sp-1) and activator protein (AP)-2 binding sites. Putative regulatory motifs for E-box, nuclear factor (NF)-1 and progesterone response element were also found by computer assisted analysis. Transient expression assays of truncated promoter-reporter constructs in HeLa cells showed that this gene is induced by phorbol esters (PDB) and cyclic-AMP-dependent protein kinase signal activation. Furthermore, the genomic organization of the PFKFB3 gene is reported. This gene spans more than 26 kb containing at least 16 exons that accounts for the two reported isoforms, inducible and ubiquitous, generated through alternative splicing of exon 15.

HERC3 binding to and regulation by ubiquitin.

Members of the HERC (domain homologous to E6 associated protein carboxy-terminus and RCC1 domain protein) family may function both as guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we identify an unstudied member, HERC3. This protein was recognized by specific antibodies in different cell types. HERC3 was located in the cytosol and in vesicular-like structures containing beta-COP, ARF and Rab5 proteins. Involvement of HERC3 in the ubiquitin system was suggested by its ability to interact with ubiquitin. The conserved cysteine in HECT proteins was not essential for this non-covalent binding. Moreover, HERC3 was a substrate of ubiquitination being degraded by the proteasome. These observations indicate a fine regulation of HERC3 and suggest a role in vesicular traffic and ubiquitin-dependent processes.

PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate.

Fructose-2,6-bisphosphate is responsible for mediating glucagon-stimulated gluconeogenesis in the liver. This discovery has led to the realization that this compound plays a significant role in directing carbohydrate fluxes in all eukaryotes. Biophysical studies of the enzyme that both synthesizes and degrades this biofactor have yielded insight into its molecular enzymology. Moreover, the metabolic role of fructose-2,6-bisphosphate has great potential in the treatment of diabetes.

Detection of mRNA encoding H(1) receptor and iNOS by RT-PCR in autoimmune myocarditis with special reference to changes in heart contractility.

Cardiac tissue from autoimmune myocarditis mice was studied to evaluate the expression and biological activity of mRNA encoding H(1) receptor and iNOS. BALB/c inbred mice were immunized with heart protein and sacrificed at 20, 45 and 50 days post immunization. Heart contractility studies and RT-PCR assays were performed. Heart from autoimmune myocarditis mice show mRNA iNOS-related dysfunction with a decrease in heart contractility. This effect was accompanied with an increase production of cyclic GMP and was improved by treating autoimmune mice with an inhibitor of iNOS activity. In addition, autoimmune myocardium expressed an active histamine H(1) receptor mRNA coupled to phospholipase C. The activation of H(1) receptor by ThEA stimulated both phosphoinositide hydrolysis and heart contractility. Normal myocardium did not expressed neither iNOS mRNA nor H(1) receptor mRNA. In conclusions: the development of autoimmune cardiac dysfunction was associated with the expression of iNOS mRNA, cyclic GMP accumulation and the expression of an active histamine H(1) receptor mRNA with increase production of inositol phosphates. These protein emergence during the course of autoimmune myocarditis may be involved a distinct compensatory mechanism operating in this disease.

Cells overexpressing fructose-2,6-bisphosphatase showed enhanced pentose phosphate pathway flux and resistance to oxidative stress.

Changes in the content of fructose-2,6-bisphosphate, a modulator of glycolytic flux, also affect other metabolic fluxes such as the non-oxidative pentose phosphate pathway. Since this is the main source of precursors for biosynthesis in proliferating cells, PFK-2/FBPase-2 has been proposed as a potential target for neoplastic treatments. Here we provide evidence that cells with a low content of fructose-2,6-bisphosphate have a lower energy status than controls, but they are also less sensitive to oxidative stress. This feature is related to the activation of the oxidative branch of the pentose phosphate pathway and the increased production of NADPH.

Overexpression of fructose 2,6-bisphosphatase decreases glycolysis and delays cell cycle progression.

The ability to overexpress 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase (PFK-2)/(FBPase-2) or a truncated form of the enzyme with only the bisphosphatase domain allowed us to analyze the relative role of the kinase and the bisphosphatase activities in regulating fructose 2,6-bisphosphate (Fru-2,6-P(2)) concentration and to elucidate their differential metabolic impact in epithelial Mv1Lu cells. The effect of overexpressing PFK-2/FBPase-2 resulted in a small increase in the kinase activity and in the activity ratio of the bifunctional enzyme, increasing Fru-2,6-P(2) levels, but these changes had no major effects on cell metabolism. In contrast, expression of the bisphosphatase domain increased the bisphosphatase activity, producing a significant decrease in Fru-2,6-P(2) concentration. The fall in the bisphosphorylated metabolite correlated with a decrease in lactate production and ATP concentration, as well as a delay in cell cycle. These results provide support for Fru-2,6-P(2) as a regulator of glycolytic flux and point out the role of glycolysis in cell cycle progression.

Fructose-1,6-bisphosphate inhibits the expression of inducible nitric oxide synthase caused by oxygen-glucose deprivation through the inhibition of glutamate release in rat forebrain slices.

Fructose-1,6-bisphosphate (FBP) is a glycolytic pathway intermediate with a neuroprotective action in animal models of brain ischaemia. We addressed the question of whether FBP acts through inhibiting inducible nitric oxide synthase (iNOS) expression via reduction of glutamate release, since we have recently demonstrated that glutamate is involved in the expression of iNOS. FBP (5 mM) added to the incubation solution of rat forebrain slices subjected to oxygen-glucose deprivation (OGD) inhibited glutamate release significantly (around 40%). FBP also inhibited the induction of the calcium-independent NOS activity and reduced the levels of iNOS protein in rat forebrain slices subjected to OGD. We conclude that the action of FBP by reducing glutamate release and iNOS expression, both of which have been implicated in cell damage, is a reason for further evaluation of FBP as a neuroprotectant.

Hepatic preconditioning preserves energy metabolism during sustained ischemia.

We evaluated the possibility that ischemic preconditioning could modify hepatic energy metabolism during ischemia. Accordingly, high-energy nucleotides and their degradation products, glycogen and glycolytic intermediates and regulatory metabolites, were compared between preconditioned and nonpreconditioned livers. Preconditioning preserved to a greater extent ATP, adenine nucleotide pool, and adenylate energy charge; the accumulation of adenine nucleosides and bases was much lower in preconditioned livers, thus reflecting slower adenine nucleotide degradation. These effects were associated with a decrease in glycogen depletion and reduced accumulation of hexose 6-phosphates and lactate. 6-Phosphofructo-2-kinase decreased in both groups, reducing the availability of fructose-2, 6-bisphosphate. Preconditioning sustained metabolite concentration at higher levels although this was not correlated with an increased glycolytic rate, suggesting that adenine nucleotides and cAMP may play the main role in the modulation of glycolytic pathway. Preconditioning attenuated the rise in cAMP and limited the accumulation of hexose 6-phosphates and lactate, probably by reducing glycogen depletion. Our results suggest the induction of metabolic arrest and/or associated metabolic downregulation as energetic cost-saving mechanisms that could be induced by preconditioning.

Interaction and functional cooperation of NF-kappa B with Smads. Transcriptional regulation of the junB promoter.

The transforming growth factor-beta (TGF-beta) family of cytokines regulates diverse cellular processes through control of the expression of target genes. Smad proteins are a recently identified family of signal transducers for members of the TGF-beta family. Smads act as transcriptional regulators through binding to DNA and interacting with a variety of transcription factors. Here, we identified a kappaB site as a TGF-beta-responsive region in the 3'-downstream junB promoter region. We also demonstrate that kappaB sites alone are sufficient to mediate immediate transcriptional activation by TGF-beta. Transactivation of kappaB sites by TGF-beta requires an intact NF-kappaB pathway, cooperates with known activators of this pathway, and is mediated by Smad family members. Furthermore, we show that Smad3 interacts with p52 in vivo. These data expand the model in which Smad proteins undergo multiple interactions with several transcription factors that could induce either activation or repression of gene expression.

Inhibition of cyclooxygenase-2 decreases DNA synthesis induced by platelet-derived growth factor in Swiss 3T3 fibroblasts.

NS-398 [N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide], a selective inhibitor of cyclooxygenase-2 (COX-2), inhibited proliferation induced by platelet-derived growth factor (PDGF) in Swiss 3T3 fibroblasts. The effect of NS-398 was found to be concentration-dependent. The half-maximal effect occurred at approximately 0.1 microM. NS-398 decreased mitogenesis at subsaturating PDGF concentrations and the inhibitory effect of NS-398 was overcome by increasing PDGF concentration. SC-236, another COX-2 selective inhibitor, also inhibited PDGF-induced proliferation. In contrast, two selective COX-1 inhibitors, valeryl salicylate and ketorolac, had no significant inhibitory effect on PDGF-stimulated DNA synthesis. The inhibition was obtained when NS-398 was added during the first hour after PDGF addition. At 1 h, PDGF induced COX-2 protein and prostaglandin (PG)E(2) synthesis, and NS-398 blocked the synthesis of PGE(2). The inhibitory effect of NS-398 on PDGF-stimulated DNA synthesis was counteracted by 280 nM PGE(2). The antimitogenic action of NS-398 and SC-236 suggests that selective inhibition of COX-2 may produce antiproliferative effects with substantial safety advantages over nonselective COX inhibitors.