Marina Macías-Silva

Determining and understanding the control of glycolysis in fast‐growth tumor cells

Control analysis of the glycolytic flux was carried out in two fast‐growth tumor cell types of human and rodent origin (HeLa and AS‐30D, respectively). Determination of the maximal velocity (Vmax) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153–306 times) and phosphfructokinase‐1 (PFK‐1) (22–56 times) had higher over‐expression in rat AS‐30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady‐state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK‐1, were increased compared with hepatocytes. In HeLa cells, Vmax values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6–9 times for both hexokinase and PFK‐1). Elasticity‐based analysis of the glycolytic flux in AS‐30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK‐1, and the Glc6P branches) exerted most of the flux control (70–75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P‐producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK‐1. Kinetic analysis of PFK‐1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast‐growth tumor cells was still controlled by an over‐produced, but Glc6P‐inhibited hexokinase.

Species heterogeneity of hepatic α1-adrenoceptors : α1A-, α1B- and α1C-subtypes

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.

Angiotensin II stimulates phosphoinositide turnover and phosphorylase through AII-1 receptors in isolated rat hepatocytes

Angiotensin II stimulated the activity of phosphorylase a (EC50 approximately 3 nM). The effect of two receptor subtype-selective nonpeptide antagonists, DuP 753 (AII-1 selective) and PD123177 (AII-2 selective), was studied. It was observed that DuP 753 inhibited the effect of angiotensin II (IC50 100 nM) but in contrast, PD123177 was without effect on this action of the peptide hormone. Angiotensin II stimulated the labeling of phosphatidylinositol (resynthesis) and the release of inositol phosphates (breakdown). These effects of angiotensin II were blocked by DuP 753 but not by PD123177. The antagonists were without effect by themselves on these parameters. The results clearly indicate that angiotensin II receptors of the AII-1 subtype are coupled to phosphoinositide turnover and mediate phosphorylase activation in isolated rat hepatocytes.

Vesicular Trafficking and Signaling for Cytokine and Chemokine Secretion in Mast Cells

Upon activation mast cells (MCs) secrete numerous inflammatory compounds stored in their cytoplasmic secretory granules by a process called anaphylactic degranulation, which is responsible for type I hypersensitivity responses. Prestored mediators include histamine and MC proteases but also some cytokines and growth factors making them available within minutes for a maximal biological effect. Degranulation is followed by the de novo synthesis of lipid mediators such as prostaglandins and leukotrienes as well as a vast array of cytokines, chemokines, and growth factors, which are responsible for late phase inflammatory responses. While lipid mediators diffuse freely out of the cell through lipid bilayers, both anaphylactic degranulation and secretion of cytokines, chemokines, and growth factors depends on highly regulated vesicular trafficking steps that occur along the secretory pathway starting with the translocation of proteins to the endoplasmic reticulum. Vesicular trafficking in MCs also intersects with endocytic routes, notably to form specialized cytoplasmic granules called secretory lysosomes. Some of the mediators like histamine reach granules via specific vesicular monoamine transporters directly from the cytoplasm. In this review, we try to summarize the available data on granule biogenesis and signaling events that coordinate the complex steps that lead to the release of the inflammatory mediators from the various vesicular carriers in MCs.

Inhibitory Smad7: Emerging Roles in Health and Disease

Smad7 is an inhibitory Smad protein that blocks Transforming Growth Factor-beta (TGF-β) signaling through a negative feedback loop, also capable of mediating the crosstalk between TGF-β and other signaling pathways. Smad7 mRNA and protein levels are upregulated after TGF-β signaling; subsequently, Smad7 protein binds TGF-β type I receptor blocking R-Smad phosphorylation and eventually TGF-β signaling. Because of this inhibitory function, Smad7 can antagonize diverse cellular processes regulated by TGF-β such as cell proliferation, differentiation, apoptosis, adhesion and migration. Smad7 induction by different cytokines, besides TGF-β, is also critical for crosstalk/integration of a variety of signaling pathways, and relevant in the pathology of some diseases. Thus, Smad7 plays a key role in the control of various physiological events, and even in some pathological processes including fibrosis and cancer. This review highlights the main known functions of Smad7 with a particular focus on the relevance that alterations of Smad7 function may have in homeostasis, also describing some Smad7 emerging roles in the development of several human diseases that identify this protein as a potential therapeutic target.

RETRACTED: 6-OHDA-induced apoptosis and mitochondrial dysfunction are mediated by early modulation of intracellular signals and interaction of Nrf2 and NF-κB factors

6-Hydroxydopamine (6-OHDA) is a neurotoxin that generates an experimental model of Parkinsons disease in rodents and is commonly employed to induce a lesion in dopaminergic pathways. The characterization of those molecular mechanisms linked to 6-OHDA-induced early toxicity is needed to better understand the cellular events further leading to neurodegeneration. The present work explored how 6-OHDA triggers early downstream signaling pathways that activate neurotoxicity in the rat striatum. Mitochondrial function, caspases-dependent apoptosis, kinases signaling (Akt, ERK 1/2, SAP/JNK and p38) and crosstalk between nuclear factor kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) were evaluated at early times post-lesion. We found that 6-OHDA initiates cell damage via mitochondrial complex I inhibition, cytochrome c and apoptosis-inducing factor (AIF) release, as well as activation of caspases 9 and 3 to induce apoptosis, kinase signaling modulation and NF-κB-mediated inflammatory responses, accompanied by inhibition of antioxidant systems regulated by the Nrf2 pathway. Our results suggest that kinases SAP/JNK and p38 up-regulation may play a role in the early stages of 6-OHDA toxicity to trigger intrinsic pathways for apoptosis and enhanced NF-κB activation. In turn, these cellular events inhibit the activation of cytoprotective mechanisms, thereby leading to a condition of general damage.

S-allyl cysteine protects against 6-hydroxydopamine-induced neurotoxicity in the rat striatum: involvement of Nrf2 transcription factor activation and modulation of signaling kinase cascades.

Pharmacological activation at the basal ganglia of the transcription factor Nrf2, guardian of redox homeostasis, holds a strong promise for the slow progression of Parkinsons disease (PD). However, a potent Nrf2 activator in the brain still must be found. In this study, we have investigated the potential use of the antioxidant compound S-allyl cysteine (SAC) in the activation of Nrf2 in 6-hydoxydopamine (6-OHDA)-intoxicated rats. In the rat striatum, SAC by itself promoted the Nrf2 dissociation of Keap-1, its nuclear translocation, the subsequent association with small MafK protein, and further binding of the Nrf2/MafK complex to ARE sequence, as well as the up-regulation of Nrf2-dependent genes encoding the antioxidant enzymes HO-1, NQO-1, GR, and SOD-1. In vivo and in vitro experiments to identify signaling pathways activated by SAC pointed to Akt as the most likely kinase participating in Nrf2 activation by SAC. In PC12 cells, SAC stimulated the activation of Akt and ERK1/2 and inhibited JNK1/2/3 activation. In the rat striatum, the SAC-induced activation of Nrf2 is likely to contribute to inhibit the toxic effects of 6-OHDA evidenced by phase 2 antioxidant enzymes up-regulation, glutathione recovery, and attenuation of reactive oxygen species (ROS), nitric oxide (NO), and lipid peroxides formation. These early protective effects correlated with the long-term preservation of the cellular redox status, the striatal dopamine (DA) and tyrosine hydroxylase (TH) levels, and the improvement of motor skills. Therefore, this study indicates that, in addition to direct scavenging actions, the activation of Nrf2 by SAC might confer neuroprotective responses through the modulation of kinase signaling pathways in rodent models of PD, and suggests that this antioxidant molecule may have a therapeutic value in this human pathology.

Inhibition of hormone-stimulated inositol phosphate production and disruption of cytoskeletal structure. Effects of okadaic acid, microcystin, chlorpromazine, W7 and nystatin

Inhibition of protein phosphatases 2A and 1 by okadaic acid and microcystin leads to cytoskeletal disruption and formation of plasma membrane blebs (blebbing) in hepatocytes. This phenomenon is associated to a marked inhibition of receptor-mediated and G-protein-mediated phosphoinositide turnover in rat hepatocytes. Other cytoskeletal-disrupting drugs such as chlorpromazine, W7 and nystatin mimic the effect of these protein phosphatase inhibitors on phosphoinositide metabolism and blebbing. Our data suggest that the coupling between G-protein and phospholipase C might be altered by cytoskeletal disruption.

Effect of okadaic acid on hormone- and mastoparan-stimulated phosphoinositide turnover in isolated rat hepatocytes

Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A which seems to be useful for identifying biological processes that are controlled by reversible phosphorylation of proteins. We report here that okadaic acid inhibits in isolated hepatocytes the stimulations of phosphoinositide turnover induced by epinephrine, angiotensin II and vasopressin. Mastoparan, a peptide toxin from wasp venom that mimics receptors by activating G-proteins, also stimulates the accumulation of inositol phosphates in hepatocytes. Interestingly, this action of mastoparan was also inhibited by okadaic acid. Our data indicate that okadaic acid inhibits the phosphoinositide turnover signal transduction system in hepatocytes at a level distal to the receptors.

Transforming growth factor-β/SMAD Target gene SKIL is negatively regulated by the transcriptional cofactor complex SNON-SMAD4.

Background: Human SKIL gene encodes for SNON, a negative regulator of the TGF-β/SMAD pathway. Results: We provide a molecular mechanism of transcriptional regulation of SKIL gene expression by TGF-β/SMADs. Conclusion: Transcriptional cofactor complex SNON-SMAD4 negatively controls the expression of SKIL gene. Significance: The formation and function of complex SNON-SMAD4 are impaired in cancer cells lacking SMAD4, which affects TGF β-target gene regulation. The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified.